Methods for treatment of insulin-like growth factor-1 (igf-1) deficiency

ABSTRACT

The present invention provides methods and compositions for increasing the growth rates, alleviating the symptoms, or improving the metabolism of human patients having insulin-like growth factor-I deficiency (IGFD). The invention relates to methods comprising administering insulin-like growth factor-I to a patient having a height which, at the time of treatment or prior to initial treatment with IGF-1, is at least about 2 standard deviations below normal for a subject of the same age and gender, a blood level of insulin-like growth factor-I that, and at the time of treatment or prior to initial treatment with IGF-1, is below normal mean levels, usually at least about 1 standard deviations below normal mean levels, for age and gender.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/502,579, filed Sep. 12, 2003, which application is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for increasingthe growth rates, alleviating the symptoms, or improving the metabolismof human patients having insulin-like growth factor-1 deficiency.

BACKGROUND OF THE INVENTION

The American Academy of Pediatrics and the American Academy of ClinicalEndocrinology define short stature based on height as more than twostandard deviations below the average population height. A child withshort stature is shorter than 97.5% of children of a similar age andgender and typically attains final adult heights of no more thanapproximately 5′4″ for boys and 4′11″ for girls. It is estimated that380,000 children in the U.S. with short statue are referred to pediatricendocrinologists for evaluation.

Children with short stature who are referred for evaluation and possibletreatment continue to pose a dilemma for specialists despite decades ofdedicated research. For patients with no demonstrable cause for theirgrowth failure, a workup usually ensues which first seeks todifferentiate between normal variation, in which the child should reachan adult height concordant with that of his family, and pathologicconditions. In cases of marked short stature, in which the predictedadult height is also low, it often becomes necessary to test the statusof the growth hormone (GH)-insulin-like growth factor (TGF) axis.

Patients with abnormalities in the GH-IGF axis have a number of possibleetiologies. They can present with GH deficiency (GHD), at timesattributable to congenital or acquired central nervous system (CNS)lesions affecting the hypothalamus or pituitary, which is almostinvariably accompanied by low IGF-1 levels in children. Alternatively,they can present “primary IGF deficiency” associated with low IGF-1levels in the face of seemingly normal GH secretion, Because IGF-1 is anessential mediator of GH's statural effects, primary IGF deficiency canhave similar clinical outcomes to GH deficiency. Such cases of primaryIGF deficiency, in otherwise healthy and well-nourished patients, arelikely to be caused by a defect somewhere in the GH-IGF axis downstreamfrom the secretion of GH. This type of GH insensitivity is as yetunexplained in most cases, although it has been associated withmutations affecting the extra-cellular domain of the GH receptor in 1-5%of idiopathic short stature (ISS) children and adults, with mutations inStat5b, with mutations in the acid labile subunit (ALS), or withmutations or polymorphisms in the IGF-1 gene itself.

GH deficiency is well recognized as a disease requiring replacementtherapy with GH for short stature and in adults for body composition,bone density, cardiac function and for well being. By contrast, low IGFlevels, in the presence of normal GH secretion, has been previouslyusually associated only with a rare disease, recognized as Laronsyndrome or growth hormone insensitivity syndrome (GHIS).

Most patients with Laron syndrome or &HIS lack growth hormone receptorbinding activity and have absent or very low GM-binding protein (GiHBP)activity in blood. Such patients have a mean height standard deviationscore (SDS) of about −5 to −6, are resistant to GH treatment, and haveincreased serum concentrations of GH and low serum concentrations ofinsulin-like growth factor (IGF-1). As children they show a staturalgrowth response to treatment with IGF-1.

The disease of short stature due to partial GH receptor defects wastraditionally seen as primarily a disease characterized by a low GHBPlevel rather than a low IGF-1 level, with IGF-1 levels being only at thelow end of the normal range. Specifically, the patient is defined ashaving a height of at least about 2 standard deviations or more belowthe normal mean for a corresponding age and gender (at least −2.0 SDbelow the mean), a serum level of high-affinity growth hormone bindingprotein that is at least 2 standard deviations below normal mean levels,a serum-level of IGF-1 that is below normal mean levels, and a serumlevel of growth hormone that is at least normal.

The importance of this classification of the various factors affectingshort stature is shown in the relative numbers of patients who are: 1)IGF-1 deficient and GH deficient and 2) IGF-1 deficient and GHsufficient. Current literature would predict that many more children andadults would be IGF-1 deficient due to GH deficiency than would be IGF-1deficient and GH sufficient.

Unlike GH deficiency (CHD), IGF-1 deficiency (IGFD) has not beenrecognized or appreciated as a disease with endocrine origins and inneed of replacement therapy. Thus, there remains a need in the art formethods of treatment of IGF-1 deficient children and adults who do nothave Laron syndrome or partial growth hormone insensitivity syndrome.

The present invention addresses these needs.

Literature

Literature of interest includes: U.S. Pat. No. 5,824,642; Salmon W D Jr.et at, 1957, Lab Olin Med, 49:825-36; Liu, J-L and LeRoith, D, 1999,Endocrinology 140:5178-84; Lupu, F et al., 2001, Dev Biol 229:141-62;Zhou, Y et al., 1997, Proc Natl Acad Sci USA 94:13215-20; and Juul,2003, GH and IGF Research 13: 113-170. Van Wyk J J. The Somatomedins:biological actions and physiological control mechanisms in HormonalProteins and Peptides, ed C H Li, 12:81-175, Orlando, Fla.: AcademicPress; Clemmons D R et al., 1984, Clin Endocrinol Metab 13:113-43;Clemmons D R et al., 1979, N Engl J Med 301:1138-42; Clemmons D R etal., 1986, Olin Endocrinol Metal 15:629-51); Liu, J-L and LeRoith, D,1999, Endocrinology 140:5178-84; Lupu, F et al., 2001, Dev Biol229:141-62; Zhou; Y et al., 1997, Proc Natl Acad Sci USA 94:13215-20).

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for increasingthe growth rates, alleviating the symptoms, or improving the metabolismof human patients having insulin-like growth factor-1 deficiency (IGFD).The invention relates to methods comprising administering insulin-likegrowth factor-1 to a patient having a height which, at the time oftreatment or prior to initial treatment with IGF-1, is at least about 2standard deviations below a normal mean for a corresponding age andgender, a blood level of IGF-1 that, and at the time of treatment orprior to initial treatment with IGF-1, is below normal mean levels,usually at least about 1 standard deviations below normal mean levelsfor a corresponding age and gender.

The present invention is based, in part, on the discovery of a patientpopulation that can benefit from IGF-1 supplementation. Such patientsare identified as having low IGF-1 blood levels, i.e., blood levels ofIGF-1 below normal mean levels, herein described as IGF-1 deficient(“IGFD”). The present invention establishes that short stature is morecommonly related to a low IGF-1 level than it is associated with a lowGH secretion. In addition, short stature correlates better with a lowIGF-1 level than a low GHBP level. Just as standard deviation scores(SDS) are used by physicians to characterize height, an IGF-1 standarddeviation score (IGF-1 SDS) indicates how many standard deviations aperson's IGF-1 level is from the average level of the population of asimilar age and gender. Further, it has been discovered that asignificant number of children with extreme or severe short stature (−3SDS for height) have at least normal GH secretion yet are very IGFdeficient in that they have IGF-1 levels that are −3 SDS scores or less.These patients are characterized as suffering from severe primary IGFD.

Accordingly, in one aspect the invention features a method for treatinga subject having insulin-like growth factor-1 deficiency (IGFD)comprising administering to a human pediatric subject an effectiveamount of insulin like growth factor-1 (IGF-1), wherein the subject ischaracterized as follows: a) at the time of treatment or prior toinitial treatment with IGF-1, has or had a height at least about 2standard deviations (SD) below a normal mean for a corresponding age andgender, and b) at the time of treatment or prior to initial treatmentwith IGF-1, has or had a blood level of IGF-1 at least about −1 SD belownormal mean levels; wherein the subject does not have Laron syndrome orpartial growth hormone insensitivity syndrome, and wherein saidadministering is effective to treat IGFD in the subject. In relatedembodiments, said administering alleviates at least one symptom of IGFD.In further related embodiments, said administering provides for anincrease in growth rate or height.

In another aspect, the invention features a method for treating asubject having insulin-like growth factor-1 deficiency (IGFD) comprisingadministering to a human adult subject an effective amount of insulinlike growth factor-1 (IGF-1), wherein the subject is characterized asfollows: a) at the time of treatment or prior to initial treatment withIGF-1, has or had a height at least about 2 standard deviations (SD)below a normal mean for a corresponding age and gender, and 2) at thetime of treatment or prior to initial treatment with IGF-1, has or had ablood level of IGF-1 at least about −1 SD below normal mean levels;wherein the subject does not have Laron syndrome or partial growthhormone insensitivity syndrome, and wherein said administering providesfor treatment of IGFD in the subject. In related embodiments, saidadministering alleviates at least one symptom of IGFD.

In yet another aspect, the invention features a method for achieving atleast normal insulin-like growth factor-1 (IGF-1) levels for age andgender (e.g., at least or greater than −2 SD below normal mean levels,or within a range of about −2.0 to +2.0 SD from a normal mean) in aninsulin-like growth factor-1 deficiency (IGFD) subject, comprisingadministering an effective amount of insulin-like growth factor (IGF-1)to the patient, wherein the patient is characterized as follows: a)subject, at the time of treatment or prior to initial treatment withIGF-1, has or had a height at least about 2 standard deviations (SD)below a normal mean for a corresponding age and gender, and b) thesubject, at the time of treatment or prior to initial treatment withIGF-1, has or had a blood level of IGF-1 at least about −1 SD belownormal mean levels; wherein the subject does not have Laron syndrome orpartial growth hormone insensitivity syndrome, and wherein saidadministering achieves blood IGF-1 levels within a normal range for acorresponding age and gender in the subject.

In embodiments related to each of the above aspects of the invention,the subject is further characterized as having at least normal bloodlevels of growth hormone binding protein (GHBP) (e.g., within a range ofabout −2.0 to about +2.0 SD from a normal mean). In further relatedembodiments, the subject is further characterized as having a bloodlevel of growth hormone (GH) which is at least normal. In still otherembodiments, the subject has a blood level of IGF-1 that is at leastabout 2.0 SD below normal mean levels.

In one embodiment of particular interest, IGF-1 is administered in adose of about 20 to 240 μg/kg/day, which IGF-1 can be administeredsubcutaneously.

In yet other aspects the invention features a method for treating asubject having a primary insulin-like growth factor-1 deficiency (IGFD)comprising administering to a human subject having primary insulin-likegrowth factor-1 deficiency (IGFD) an effective amount of insulin likegrowth factor-1 (IGF-1), wherein the subject is characterized asfollows: a) at the time of treatment or prior to initial treatment withIGF-1, has or had a height at least about 2 standard deviations (SD)below a normal mean for a corresponding age and gender, b) the time oftreatment or prior to initial treatment with IGF-1, has or had a bloodlevel of IGF-1 at least about −1 SD below normal mean levels, and c) ata blood level of growth hormone (GH) which is at least normal, whereinthe subject does not have Laron syndrome or partial growth hormoneinsensitivity syndrome, and wherein said administering provides fortreatment of IGFD in the subject.

In still other aspects the invention features a method for achieving atleast normal insulin-like growth factor-1 (IGF-1) levels for acorresponding age and gender (e.g., within the normal range of IGF-1levels for a corresponding age and gender) in a primary insulin-likegrowth factor-1 deficiency (IGFD) subject, comprising administering aneffective amount of insulin-like growth factor (IGF-1) to a humansubject, wherein the patient is characterized as follows: a) thesubject, at the time of treatment or prior to initial treatment withIGF-1, has or had a height at least about 2 standard deviations (SD)below the normal mean for a corresponding age and gender, b) thesubject, at the time of treatment or prior to initial treatment withIGF-1, has or had a blood level of IGF-1 at least about −1 SD belownormal mean levels, and c) that subject has a blood level of growthhormone (GH) which is at least normal; wherein the subject does not haveLaron syndrome or partial growth hormone insensitivity syndrome, whereinsaid administering achieves normal blood IGF-1 levels (e.g., within thenormal range) for a corresponding age and gender in the subject.

In embodiments related to the above aspects of the invention, thesubject is further characterized as having at least normal blood levelof growth hormone binding protein (GHBP). In still other embodiments,the subject has a blood level of IGF-1 that is at least about 2.0 SDbelow normal mean levels. In one embodiment of particular interest,IGF-1 is administered in a dose of about 20 to 240 μg/kg/day, whichIGF-1 can be administered subcutaneously. In further relatedembodiments, said administering alleviates at least one symptom of IGFD.In still further related embodiments, the subject is a human pediatricsubject and said administering provides for an increase in growth rateor height.

The present invention thus also encompasses methods for treating apatient with short stature having a blood level of IGF-1, which a thetime of treatment or prior to initial treatment, is at least about 1standard deviation (SD) below normal mean levels (usually greater than 1SD below normal mean levels, with at least about 2.0 SD below normalmean levels being of particular interest); and a height which; at thetime of treatment, or prior to initial treatment, is at least about 2standard deviations (SD) below the normal mean for a corresponding ageand gender. Without being bound by any theory, administration of IGF-1increases the blood levels of IGF-1. In the case of a patient with IGFD,the methods have application where the patient does not have Laronsyndrome or partial growth hormone insensitivity syndrome.

In related embodiments, the patient also has a blood level of growthhormone binding protein (GHBP) (e.g., mean or maximal) that is at leastnormal. In further related embodiments the patient also has a bloodlevel of growth hormone (e.g., mean or maximum stimulated) which is atleast normal. The administration of IGF-1 results in alleviating asymptom associated with IGFD, which include lipid abnormalities,decreased bone density, obesity, insulin resistance, decreased cardiacperformance, decreased muscle mass, decreased exercise tolerance.Alleviation of such symptoms is of particular interest in adults. Wherethe IGFD patient is a child, of particular interest is administration ofIGF-1 to provide for an increase in the patient's height and growthrate.

Accordingly, in one aspect the invention provides a method forincreasing the growth rate of a human subject (usually a pediatricsubject) having primary IGFD comprising administering an effectiveamount of IGF-1 to said subject, whereby said subject has a heightwhich, at the time of treatment or prior to initial treatment withIGF-1, is at least about 2 standard deviations (SD) below the normalmean for a corresponding age and gender, has a blood level of IGF-1 thatat the time of treatment or prior to initial treatment with IGF-1, isgreater than 1 SD below normal mean levels, wherein the subject does nothave Laron syndrome or partial growth hormone insensitivity syndrome,and wherein said administering is effective to increase growth rate ofthe subject. In related embodiments, the subject also has a mean ormaximum stimulated blood level of growth hormone which is at leastnormal and/or at least normal blood levels of growth hormone bindingprotein. The invention is useful in the treating children of shortstature to accelerate their growth to increase their height.

In another aspect, the invention provides a method for treating IGFD inan adult patient comprising administering an effective amount of IGF-1to said patient, wherein said patient has a height which, at the time oftreatment or prior to initial treatment with IGF-1, is at least about 2SD below the normal mean for a corresponding age and gender, has a bloodlevel of IGF-1 that, at the time of treatment or prior to initialtreatment with IGF-1, is greater than 1 SD below normal mean levels, andhas a mean or maximum stimulated level of growth hormone which is atleast normal. In this aspect, the invention is useful in adults toalleviate the symptoms of their IGF deficiency.

In certain embodiments, the patient has a blood level of IGF-1 of atleast −1.0 SD, at least 2.0 SD below normal mean levels.

In certain embodiments, the invention provides methods for increasingthe growth rate or reducing the metabolic effects of IGF deficiency of apatient by administration of an effective amount of IGF-1 at 20 to 240μg/kg/day. In certain embodiments, the IGF-1 is administeredsubcutaneously.

DEFINITIONS

Before describing the invention in greater detail, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used to describe the invention herein.

As used herein, “patient” refers to any mammal, including humans,bovines, ovines, porcines, canines and felines, in need of treatment. Incertain embodiments, the patient is a human. In general, the methods ofthe invention are applicable to pediatric and adult patients.

As used herein, “insulin-like growth factor-1 deficiency”, “IGF-1deficiency”, or “IGFD” refer to a condition associated with thefollowing characteristics, a height of at least about 2 standarddeviations (SD) below the normal mean level for the corresponding ageand gender, a blood level of IGF-1 that is at least 1 SD below normalmean levels. In general, IGFD can be due to a resistance to OH action oras a result of OH deficiency (GHD). IGFD that is due to resistance to GHaction is termed primary IGFD, while IGFD resulting from GHD is termedsecondary IGFD. Primary IGFD is distinguished from secondary IGFD inthat primary IGFD is associated with at least normal GH blood levels,while secondary IGFD is associated with low blood levels of GH.

Thus, primary IGFD refers to a condition associated with the followingcharacteristics, a height of at least about 2 standard deviations (SD)below the normal mean for the corresponding age and gender, a bloodlevel of IGF-1 that is below normal mean levels, and a mean or maximumstimulated blood level of growth hormone (GH) that is at least normal(e.g., normal GH blood levels or greater than normal GH blood levels).GHBP levels are generally within the normal range.

Pediatric primary IGFD refers to pediatric patients with IGFD, whileAdult primary IGFD refers to adult patients with IGFD. Adult primaryIGFD, is similar to pediatric primary IGFD and is associated with aheight of at least 2 SD below the normal mean for the corresponding ageand gender, a blood level of IGF-1 that is at least 2 SD below thenormal mean for the corresponding age and gender, and normal growthhormone levels. Adult primary IGFD patients have increased bloodpressure, decreased cardiac performance, cardiac disease, renal diseaseimpaired exercise performance, decreased muscle mass, decreased bonedensity, obesity and abnormalities of carbohydrate and lipid metabolism.Pediatric patients with primary IGFD are capable of having their heightor growth rate increased, while adult patients are no longer capable ofachieving a greater height. In certain embodiments, the subject methodsdo not encompass treating pediatric primary IGFD patients who have ablood level of high affinity growth hormone binding protein that is atleast 2SDs below normal mean levels and do not have Laron syndrome.

The term “concentration in blood”, such as in the phrases “IGF-1concentration in blood” or “IGFBP-3 concentration in blood”, refers to aconcentration of an agent (e.g., IGF-1 or IGFBP-3) obtained in wholeblood or in a fluid obtained from blood, such as plasma or serum.

As used herein, “short stature” means a subject who has a heightstandard deviation score of about ≦2 SD below that of the normal meanfor an individual of the same age and gender.

As used herein, the term “Laron syndrome” refers to a patient exhibitingcomplete lack of growth hormone receptor (GHR) function or completegrowth hormone insensitivity syndrome (GHIS). Such patients have a meanheight standard deviation score (SDS) of about −5 to −6 and respond totreatment with IGF-1. In patients with defects in the extracellulardomain of the GHR, the lack of functional GHBP in the circulation canserve as a marker for the GH insensitivity. Additional common symptomsof “Laron syndrome” include small face and jaw, depressed nasal bridge,frontal bossing, obesity, high-pitched voice, and hypoglycemia in earlychildhood. Biochemically, Laron syndrome patients are characterized byhaving increased blood concentrations of GH and low blood GHBPconcentrations, but low blood concentrations of IGF-1.

As used herein, “partial growth hormone insensitivity syndrome”, or“partial GHIS” refers to a syndrome wherein the patient responds to thesame doses of GH as that given to GH-deficient patients, but does notrespond as well. This syndrome is further characterized in that thepatient has a height of at least about 2 standard deviations below thenormal mean for a corresponding age and gender, preferably in the rangeof about 2 to about 4 standard deviations or more below the normal meanfor a corresponding age and gender (e.g., a SD of −2.0 or −4.0), has ablood level of high-affinity GHBP that is at least 2 standard deviations(typically about 2 to about 4 standard deviations) below the normal meanlevel for humans, has a blood level of IGF-1 that is below the normalmean level for humans, and has a mean or maximum stimulated blood levelof GH that is at least normal. Mean blood levels are the mean ofmeasurements in the patient.

As used herein, “IGF-1” refers to insulin-like growth factor-1 from anyspecies, including bovine, ovine, porcine, equine, avian, and preferablyhuman, in native-sequence or in variant form, and from any source,whether natural, synthetic, or recombinant.

Suitable for use in the subject methods is human native-sequence, matureIGF-1, for example, without an N-terminal methionine, prepared, e.g., bythe process described in EP 230,869 published Aug. 5, 1987; EP 128,733published Dec. 19, 1984; or EP 288,451 published Oct. 26, 1988. Morepreferably, this native-sequence IGF-1 is recombinantly produced and isavailable for clinical investigations (see, e.g., EP 123,228 and128,733). The term “rhIGF-1” refers to recombinant human IGF-1.

As used herein, reference to “variants” or “analogs, homologs andmimics” of IGF-1 embraces compounds which differ from the structure ofnative IGF-1 by as little as the replacement and/or deletion of one ormore residues thereof, to compounds which have no apparent structuralsimilarity. Such compounds in all instances, however, have substantiallythe same activity as native IGF-1. Thus, “analogs” refers to compoundshaving the same basic structure as IGF-1, but differing in severalresidues; “homologs” refers to compounds which differ from native IGF-1by the deletion and/or replacement of a limited number of residues; and“mimics” refers to compounds which have no specific structuralsimilarity with respect to IGF-1 (indeed, a mimic need not even be apolypeptide), but such compound will display the biological activitycharacteristic of IGF-1 and/or stimulate endogenous IGF-1 production bythe body or increase the amount of endogenous IGF-1 available to bind toIGF-1 receptors.

Suitable for use in the present invention are IGF-1 variants describedin U.S. Pat. Nos. 5,077,276 issued Dec. 31, 1991; 5,164,370; 5,470,828;in PCT WO 87/01038 published Feb. 26, 1987 and in PCT WO 89/05822published Jun. 29, 1989, i.e., those wherein at least the glutamic acidresidue is absent at position 3 from the N-terminus of the maturemolecule or those having a deletion of up to five amino acids at theN-terminus. The most preferred variant has the first three amino acidsfrom the N-terminus deleted (variously designated as brain IGF, tIGF-1,des(1-3)-IGF-1, or des-IGF-1). Other compounds are the IGF-1 displacerscompounds as described below, and in U.S. Pat. Nos. 6,121,416,6,251,865, and 6,420,518.

As used herein, an “IGF binding protein” or “IGFBP”, refers to a proteinor polypeptide normally associated with or bound or complexed to IGF-1or IGF-2, whether or not it is circulatory (i.e., in blood (e.g., serum)or tissue). Such binding proteins do not include receptors. Thisdefinition includes IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5,IGFBP-6, Mac 25 (IGFBP-7), and prostacyclin-stimulating factor (PSF) orendothelial cell-specific molecule (ESM-1), as well as other proteinswith high homology to IGFBPs. Mac 25 is described, for example, inSwisshelm et al., 1995, Proc Natl Acad Sci USA, 92: 4472-4476 and Oh etal., J Biol Chem, 271: 30322-30325 (1996). PSF is described in Yamauchiet al., 1994, Biochem J, 303:591-598. ESM-1 is described in Lassalle etal., 1996, Biol Chem, 271: 20458-20464. For other identified IGFBPs,see, e.g., EP 375,438 published Jun. 27, 1990; EP 369,943 published May23, 1990; WO 89/09268 published Oct. 5, 1989; Wood et al., 1988, MolEndocrinol, 2: 1176-1185; Brinkman et al., 1988, EMBO J, 7: 2417-2423;Lee et al., 1988, Mol Endocrinol, 2:404-411; Brewer et al., 1988,Biochem Biophys Res Comm, 152: 1289-1297; EP 294,021 published Dec. 7,1988; Baxter et al., 1987, Biochem Biophys Res Comm, 147: 408-415; Leunget al., 1987, Nature, 330: 537-543; Martin et al., 1986, J Biol Chem,261:8754-8760; Baxter et al., 1988, Comp Biochem Physiol, 91B: 229-235;WO 89/08667 published Sep. 21, 1989; WO 89/09792 published Oct. 19,1989; and Binkert et al., 1989, EMBO J, 8: 2497-2502.

As used herein, “active”, “bioactive”, “biologically active” or “free”IGF-1 in the context of changing blood and tissue levels of endogenousIGF-1 refers to IGF-1 that binds to an IGF receptor or an insulinreceptor, or a hybrid IGF/insulin receptor, or to an IGF bindingprotein, or otherwise causes a biological activity of endogenous orexogenous IGF-1 to occur.

As used herein, “high-affinity growth hormone binding protein” or“high-affinity GHBP” refers to the extracellular domain of the GHR thatcirculates in blood and functions as a GHBP in several species (Ymer etal., 1985, Mol. Cell. Endocrinol. 41:153; Smith et al., 1988,Endocrinology 123:1489-1494; Emtner et al., 1990, Acta Endocrinologica(Copenh.), 122:296-302), including man (Baumann et al., 1986, J. Clin.Endocrinol. Metab., 62:134-141; EP 366,710 published 9 May 1990;Herington et al., 1986, J. Clin. Invest., 77:1817-1823; Leung et al.,1987, Nature 330:537-543. A second BP with lower affinity for GH hasalso been described that appears to be structurally unrelated to the GHR(Baumann et al., 1990, J. Clin. Endocrinol. Metab. 70:680-686, Variousmethods exist for measuring functional GHBP in blood, with the preferredmethod being a ligand-mediated immunofunctional assay (LIFA) describedby Carlsson et al. (1991, J. Clin. Endocrinol. Metab. 73:1216) and U.S.Pat. No. 5,210,017.

As used herein, “increasing the growth rate of a patient” includes notonly the situation where the patient attains a similar ultimate heightas GH-deficient patients treated with GH (i.e., patients diagnosed withGHD) or IGF-1 deficient patients treated with IGF-1, but also refers toa situation where the patient catches up in height at a similar growthrate as GH-deficient patients treated with GH or IGF-1 deficientpatients treated with IGF-1, or achieves adult height that is close tothe target height range, i.e., an ultimate height more consistent withtheir genetic potential as determined by the mid-parental target height.

As used herein, “alleviating a symptom of IGFD” refers to achieving atherapeutic benefit for a symptom associated with IGF-1 deficiency.Symptoms of IGFD patients include, but are not limited to, deincreasedgrowth rate and height, increased blood pressure, decreased cardiacperformance, cardiac disease, renal disease, neurological disease,impaired exercise performance, decreased muscle mass, decreased bonedensity, obesity and abnormalities of carbohydrate and lipid metabolism.Thus, alleviating symptoms of IGFD results in increased growth rates andheight, bone density, bone structure, improved renal and cardiacfunction, and improved glucose control and body composition.

As used herein, “treatment” or “treating” refers to inhibiting theprogression of a disease or disorder, e.g., short stature or IGFD, ordelaying the onset of a disease or disorder, e.g., short stature orIGFD, whether physically, e.g., stabilization of a discernible symptom,physiologically, e.g., stabilization of a physical parameter, or both.As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or condition, or a symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease ordisorder and/or adverse affect attributable to the disease or disorder.“Treatment,” as used herein, covers any treatment of a disease ordisorder in a mammal, such as a human, and includes: decreasing the riskof death due to the disease; preventing the disease of disorder fromoccurring in a subject which may be predisposed to the disease but hasnot yet been diagnosed as having it; inhibiting the disease or disorder,i.e., arresting its development (e.g., reducing the rate of diseaseprogression); and relieving the disease, i.e., causing regression of thedisease. Therapeutic benefits of the present invention include, but arenot necessarily limited to, reduction of risk of onset or severity ofdisease or conditions associated with short stature or IGFD.

As used herein, a “therapeutically effective amount” refers to thatamount of the compound sufficient to treat or manage a disease ordisorder, e.g., short stature or IGFD. A therapeutically effectiveamount may refer to the amount of a compound that provides a therapeuticbenefit in the treatment or management of a disease or disorder.Further, a therapeutically effective amount with respect to a compoundof the invention means that amount of compound alone, or in combinationwith other therapies, that provides a therapeutic benefit in thetreatment or management of a disease or disorder. The term can encompassan amount that improves overall therapy, reduces or avoids unwantedeffects, or enhances the therapeutic efficacy of or synergies withanother therapeutic agent.

As used herein, a “pharmaceutical composition” is meant to encompass acomposition suitable for administration to a subject such as a mammal,especially a human. In general a “pharmaceutical composition” issterile, and preferably free of contaminants that are capable ofeliciting an undesirable response within the subject (e.g., thecompound(s) in the pharmaceutical composition is pharmaceutical grade).Pharmaceutical compositions can be designed for administration tosubjects or patients in need thereof via a number of different routes ofadministration including oral, buccal, rectal, parenteral,intraperitoneal, intradermal, intracheal and the like. In someembodiments the composition is suitable for administration by atransdermal route, using a penetration enhancer other than DMSO. Inother embodiments, the pharmaceutical compositions are suitable foradministration by a route other than transdermal administration.

As used herein, the phrase “pharmaceutically acceptable carrier” refersto a carrier medium that does not interfere with the effectiveness ofthe biological activity of the active ingredient. Said carrier medium isessentially chemically inert and nontoxic.

As used herein, the phrase “pharmaceutically acceptable” means approvedby a regulatory agency of the Federal government or a state government,or listed in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly for use inhumans.

As used herein, the term “carrier” refers to a diluent, adjuvant,excipient, or vehicle with which the therapeutic is administered. Suchcarriers can be sterile liquids, such as saline solutions in water, oroils, including those of petroleum, animal, vegetable or syntheticorigin, such as peanut oil, soybean oil, mineral oil, sesame oil and thelike. A saline solution is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The carrier, if desired, can also containminor amounts of wetting or emulsifying agents, or pH buffering agents.These pharmaceutical compositions can take the form of solutions,suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like. The composition can beformulated as a suppository, with traditional binders and carriers suchas triglycerides. Examples of suitable pharmaceutical carriers aredescribed in Remington's Pharmaceutical Sciences by E. W. Martin.Examples of suitable pharmaceutical carriers are a variety of cationicpolyamines and lipids, including, but not limited toN-(1(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA) anddiolesylphosphotidylethanolamine (DOPE). Liposomes are suitable carriersfor gene therapy uses of the invention. Such pharmaceutical compositionsshould contain a therapeutically effective amount of the compound,together with a suitable amount of carrier so as to provide the form forproper administration to the subject. The formulation should suit themode of administration.

As used herein, “pharmaceutically acceptable derivatives” of a compoundof the invention include salts, esters, enol ethers, enol esters,acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases,solvates, hydrates or prodrugs thereof. Such derivatives may be readilyprepared by those of skill in this art using known methods for suchderivatization. The compounds produced may be administered to animals orhumans without substantial toxic effects and either are pharmaceuticallyactive or are prodrugs.

As used herein, the phrase “pharmaceutically acceptable salts” refers tosalts prepared from pharmaceutically acceptable, essentially nontoxic,acids and bases, including inorganic and organic acids and bases.Pharmaceutically acceptable salts include those formed with free aminogroups such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with free carboxyl groupssuch as those derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

As used herein, the phrase “mean or maximum stimulated blood level ofGH” means a GH level of about 5 ng/ml in adults and about 10 ng/ml inchildren as measured by a radioimmunoassay following a GH stimulationtest wherein a compound is administered that causes the release of GH.

“In combination with” as used herein refers to uses where, for example,the first compound is administered during the entire course ofadministration of the second compound; where the first compound isadministered for a period of time that is overlapping with theadministration of the second compound, e.g. where administration of thefirst compound begins before the administration of the second compoundand the administration of the first compound ends before theadministration of the second compound ends; where the administration ofthe second compound begins before the administration of the firstcompound and the administration of the second compound ends before theadministration of the first compound ends; where the administration ofthe first compound begins before administration of the second compoundbegins and the administration of the second compound ends before theadministration of the first compound ends; where the administration ofthe second compound begins before administration of the first compoundbegins and the administration of the first compound ends before theadministration of the second compound ends. As such, “in combination”can also refer to regimen involving administration of two or morecompounds. “In combination with” as used herein also refers toadministration of two or more compounds which may be administered in thesame or different formulations, by the same of different routes, and inthe same or different dosage form type.

It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely”,“only” and the like in connection with the recitation of claim elements,or the use of a “negative” limitation.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anindividual” includes one or more individuals, and reference to “themethod” includes reference to equivalent steps and methods known tothose skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.Citation or discussion of a reference herein shall not be construed asan admission that such is prior art to the present invention.

The invention will now be described in more detail.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting a plot of Height SDS vs. Blood Level ofIGF-1 in adult patients previously characterized as suffering from Type2 diabetes mellitus.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the surprising finding that IGF-1administration increases the statural growth of certain pediatricpatient populations not previously known to be amenable to treatmentwith IGF-1 to achieve a more normal height (e.g., toward or within thenormal range for a corresponding age and gender). While not being boundby a particular theory, while the level of Growth Hormone bindingprotein (GHBP) has been found to reflect the state of the actual GHreceptor, it is not a good indicator of intracellular signaling pathways“downstream” from the event of GH receptor binding. Thus, there aresurprisingly many more pediatric patients who have evidence of GHresistance than can be identified by only measuring the level of GHBP.In view of the discoveries described herein, it is now estimated that asurprisingly large number of children, approximately 60,000 children inthe U.S. and Western Europe suffer from primary insulin-like growthfactor deficiency (IGFD). Moreover, approximately 12,000 children in theU.S. and Europe are afflicted by Severe Primary IGFD, defined aschildren who have a Height SDS of at least minus three (≦−3) below thenormal mean for a corresponding age and gender (i.e., at least 3 or moreSD below), with IGF-1 SDS of at least minus three (≦−3) below the normalmean for a corresponding age and gender (i.e., at least 3 or more SDbelow) and levels of growth hormone that are at least within the normalrange. If left untreated, these children suffering from Severe PrimaryIGFD will attain final adult heights of no more than approximately 5′1″for boys and 4′9½″ for girls.

Accordingly, a large number of adults suffer from the adverse metaboliceffects of life-long IGFD. At least 120,000 individuals in the U.S. andWestern Europe suffer from Adult Primary IGFD. Adult Primary IGFD istypically characterized by life-long IGF-1 deficiency. This disorder issimilar to Pediatric Primary IGFD and is associated with a height SDS ofat least minus two (≦−2) below the normal mean for a corresponding ageand gender (i.e., at least 2 or more SD below), IGF-1 SDS of at leastminus two (≦−2) below the normal mean (i.e., at least 2 or more SDbelow), and normal growth hormone levels. Adult IGFD patients haveincreased blood pressure, decreased cardiac performance, cardiacdisease, renal disease, impaired exercise performance, decreased musclemass, decreased bone density, obesity and abnormalities of carbohydrateand lipid metabolism. Replacement therapy with rhIGF-1 will havebeneficial effects with respect to these metabolic and functionalabnormalities.

An association between adult height and mortality from coronary heartdisease (CHD) has been detected in several studies. The very large“Nurses health study” a prospective cohort of 121,700 U.S. female nursesaged 30-55 years, showed that height is inversely related to risk ofcoronary heart disease in women (Rich-Edwards et al., 1995, Am JEpidemiol. 142:909-17). Moreover, a recent study relating short statureto clinical procedures in 1,046 men, showed that the shorter men had ahigher prevalence and greater severity of angiographically verified CUD(Nwasokwa et al, 1997, Am Heart J 133:147-52). Recently it has beenshown that short stature is an independent risk factor for coronaryheart disease (Forsen et al., 2000, J Intern Med. 248:326-32). Theseauthors speculate about, but rule out, a deficiency of growth hormone asbeing a possible cause of the original short stature and the subsequentadverse effects on the heart. In addition, these authors do notspeculate as to the possibility that IGFD might be the cause of theshort stature and the coronary heart disease in these patients withshort stature.

Another large study in Europe showed that short stature is associatedwith several metabolic disorders and that skeletal disproportion isassociated with diabetes in men while confirming the association ofshort stature with coronary heart disease in women (Han et al., 1997,Eur J Clin Nutr. 51:804-9).

Furthermore, there is also a relationship between short stature andrenal disease. A recent study measured the level of albumin in urine of3,960 patients who were 40 years old and older (Metcalf et al., 1997,Int Journal of Obesity 21: 203-210). Microalbuminuria was defined asbeing present if there was greater than 28 mg/dl of albumin in the urinebased on reference value from the normal population. The height of theindividuals was also measured. Persistent microalbuminuria is predictiveof diabetic nephropathy (renal disease) and of increased morbidity andmortality from cardiovascular disease. In these patients short staturewas a significant predictor of increased urinary albumin excretion.Other studies have found a similar relationship (Gould et al., 1993, BrMed J 306:240-243). Metcalf et al. do not explain the basis for thisrelationship between height and a marker of renal disease. Because ofthe unexpected relationship described in the present specificationbetween blood IGF-1 level and height, an explanation can be provided forthese findings.

There is a large literature showing that IGF-1 affects the kidney interms of both structure and function (Clark and Roelfsema, 2001, J AmSoc Nephrol. 12:1297-306). Therefore it can be seen that the aboverelationship between height and renal disease can be explained by theblood levels of IGF-1 varying with height. Therefore patients who areshort and have low blood levels of IGF-1 (patients who are IGFD) arepatients who would benefit most from treatment with IGF-1. In these IGFDpatients replacement therapy with IGF-1 would be expected to reducemicroalbuminuria, improve renal function, and reduce mortality.

It is clear that it is not height itself that has these effects but theunderlying mechanisms that affect height. Forsen et al. state that thefactors and mechanisms through which the factors act remain unknown. Ithas been shown in obese patients and in Type 2 diabetics that overallIGF-1 blood levels are relatively normal (Frystyk et al, 1999, DiabetesMetab Res Rev. 15:314-22). However there is little information on theIGF-1 levels in adults, or in short adults with cardiovascular diseaseor heart disease.

The low IGF-1 level in the presence of levels of GH that are at leastnormal is indicative of GH resistance. This concept of growth hormoneinsensitivity syndrome (GHIS), of a low GHBP level being indicative ofGH resistance, pre-supposed that GH resistance would be associated witha low blood level of the GHBP and therefore a low number of GHreceptors. However, it is now recognized as part of this invention thatmany more patients than previously described are short due to GHresistance. This is because, as described herein, the primary measuresof GH resistance is the blood IGF-1 concentration and the blood GH levelrather than the blood level of the GHBP. Without being limited to anyone theory, GH resistance is more likely due to defects in intracellularGH signaling than to a deficit in the number or function of the GHreceptors on cells themselves.

Therefore it is clear that the GHBP level in blood is only indicative ofthe degree of GH resistance in a minority of patients. A betterindicator, or blood marker, or biochemical characteristic of a patient,of the degree of GH resistance (as seen in individuals suffering fromshort stature) is the blood IGF-1 level. Therefore, replacement therapywith IGF-1 is better gauged and administered to patients who are IGF-1deficient than those that are GHBP deficient.

The level of blood IGF-1 also has profound metabolic effects. Therefore,as children with IGFD become adults, they continue to suffer from theeffects of IGF-1 deficiency. Since after puberty the growth plates inthe long bones fuse and additional cartilage and bone growth andincrease in height can no longer occur, rhIGF-1 replacement therapy doesnot cause growth in adults. However, low levels of blood IGF-1 are alsofrequently associated with other metabolic disorders, including lipidabnormalities, decreased bone density, obesity, insulin resistance,decreased cardiac performance, decreased muscle mass, decreased exercisetolerance and well being. These disorders typically become increasinglyapparent after a prolonged period of IGF-1 deficiency, as occurs inadulthood. Accordingly, this disorder is referred to as Adult IGFD.

It is an object of the present invention to provide methods andcompositions for increasing the height and growth rates and improvingthe metabolism and function of patients with IGFD. In certainembodiments, as in the case of IGFD subjects, the goal of treatment isto restore biologically active IGF-1 levels or to increase tissueexposure to IGF-1, to those found within normal subjects of the same ageand gender, and, in children, thereby increase the heights and growthrate of these subjects to within the normal range for subjects of thesame age and gender, while, in adults, reducing the incidence of theadverse metabolic and functional defects which characterize IGFD.

Administration of IGF-1

The present invention provides methods and compositions for increasingthe height and growth rates and improving the metabolism of patientswith IGFD by administering to the patients an effective amount of IGF-1.In some embodiments, native human IGF-1 is used. In other embodiments,IGF-1 variants are used. In yet other embodiments, IGF-1 displacers areused.

Suitable for use in the subject methods are IGF-1 variants. IGF-1variants can be designed that retain efficient binding to the type I IGFreceptor, yet would have reduced binding to serum carrier proteins, e.g.IGFBPs. In one aspect, the design of these variants is based on theobservation that insulin does not bind to serum carrier proteins. SeeU.S. Pat. No. 4,876,242, issued Oct. 24, 1989, herein expresslyincorporated by reference in its entirety. Evidence from synthetic,insulin-like two chain analogs suggests that amino acids of IGF-1responsible for carrier protein binding are in the B region of IGF-1.Therefore a synthetic gene for human IGF-1 can be modified to encode anIGF-1 variant in which the first 16 amino acids of hIGF-1 are replacedby the first 17 amino acids of the B chain of human insulin. Thesynthetic gene is then placed in a yeast recombinant DNA expressionsystem and the peptide analog which is produced by the modified yeastcells is extracted therefrom and purified. Additional modifications ofthe IGF-1 molecule have been carried out leading to additional analogs,all of which have substantial IGF-1 type I receptor binding and reducedbinding to serum carrier proteins.

Other IGF-1 variants and analogs well known in the art are also suitablefor use in the subject methods. Such variants include, for example, thevariant having resides 1-69 of authentic IGF-1, further described in WO96/33216, and the two-chain IGF-1 superagonists which are derivatives ofthe naturally occurring single-chain IGF-1 having an abbreviated Cdomain, further described in EP 742,228. IGF-1 analogs are of theformula: BC^(n), A wherein B is the B domain of IGF-1 or a functionalanalog thereof, C is the C domain of IGF-1 or a functional analogthereof, n is the number of amino acids in the C domain and is fromabout 6 to about 12 amino acids, including about 8 to about 10 aminoacids, and A is the A domain of IGF-1 or a functional analog thereof.

Also suitable for use in the subject methods are functional mutants ofIGF-1 that are well known in the art. Such functional mutants includethose described in Cascieri et al. (1988, Biochemistry 27:3229-3233),which discloses four mutants of IGF-1, three of which have reducedaffinity to the Type I IGF receptor. These mutants are: (Phe²³, Phe²⁴,Tyr²⁵) IGF-1 (which is equipotent to human IGF-1 in its affinity to theTypes 1 and 2 IGF and insulin receptors), (Leu²⁴)IGF-1 and (Ser²⁴)IGF-1(which have a lower affinity than IGF-1 to the human placental Type IIGF receptor, the placental insulin receptor, and the Type I IGFreceptor of rat and mouse cells), and desoctapeptide (Leu²⁴)IGF-1 (inwhich the loss of aromaticity at position 24 is combined with thedeletion of the carboxyl-terminal D region of hIGF-1, which has loweraffinity than (Leu²⁴)IGF-1 for the Type I receptor and higher affinityfor the insulin receptor). These four mutants have normal affinities forhuman serum binding proteins.

Also suitable for use with the subject methods include structuralanalogs of IGF-1 well known in the art. Such structural analogs includethose described in Bayne et al. (1988, J Biol Chem 264:11004-11008),which discloses three structural analogs of IGF-1: (1-62)IGF-1, whichlacks the carboxyl-terminal 8-amino-acid D region of IGF-1;(1-27,Gly⁴,38-70)IGF-1, in which residues 28-37 of the C region of IGF-1are replaced by a four-residue glycine bridge; and (1-27,Gly⁴,38-62)IGF-1, with a C region glycine replacement and a D region deletion.Peterkofsky et al. (1991, Endocrinology, 128: 1769-1779) discloses datausing the Gly⁴ mutant of Bayne et al., supra. U.S. Pat. No. 5,714,460refers to using IGF-1 or a compound that increases the activeconcentration of IGF-1 to treat neural damage.

Other structural analogs include those described in Cascieri et al.(1989, J Biol Chem, 264: 2199-2202) discloses three IGF-1 analogs inwhich specific residues in the A region of IGF-1 are replaced with thecorresponding residues in the A chain of insulin. The analogs are:(Ile⁴¹, Glu⁴⁵, Gln⁴⁶, Thr⁴⁹, Ser⁵⁰, Ile⁵¹, Ser⁵³, Tyr⁵⁵, Gln⁵⁶)IGF-1, anA chain mutant in which residue 41 is changed from threonine toisoleucine and residues 42-56 of the A region are replaced;(Thr⁴⁹,Ser⁵⁰,Ile⁵¹)IGF-1; and (Tyr⁵⁵, Gln⁵⁶)IGF-1.

IGF-1 point variants which bind to IGFBP-1 or IGFBP-3, thus inhibitingthe interaction of endogenous IGF-1 with IGFBPs are also suitable foruse with the subject methods and are described in U.S. Pat. No.6,509,443.

In another embodiment, the level of IGF-1 is increased by administeringa compound that prevents or inhibits the interaction of IGF-1 with itsbinding proteins, such as a IGF-1 agonist molecules that are capable ofeffectively inhibiting the interaction of IGF-1 with its bindingproteins, thereby allowing IGF-1 to bind to the IGF receptor foractivity. Such IGF-1 agonists suitable for use in the subject methodsinclude those described in See U.S. Pat. No. 6,251,865, issued Jun. 26,2001, herein expressly incorporated by reference in its entirety. TheseIGF-1 agonist molecules can effectively displace IGF-1 bound to IGFBP.The IGF binding proteins (IGFBPs) are a family of at least six proteins(See Jones and Clemmons, 1995, Endocr Rev, 16: 3-34; Bach and Rechler,1995, Diabetes Reviews, 3: 38-61), with other related proteins alsopossibly binding the IGFs. The IGFBPs bind IGF-1 and IGF-2 with varyingaffinities and specificities. See Jones and Clemmons, supra; Bach andRechler, supra. For example, IGFBP-3 binds IGF-1 and IGF-2 with asimilar affinity, whereas IGFBP-2 and IGFBP-6 bind IGF-2 with a muchhigher affinity than they bind IGF-1. See Bach and Rechler, supra; Oh etal., 1993, Endocrinology, 132, 1337-1344.

Also suitable for use in the subject methods include binding molecules,other than a natural IGFBP, as described in WO 94/04569 than can preventthe binding of IGF-1 to a IGFBP by binding to IGF-1 and therebyenhancing the biological activity of IGF-1. In addition, other moleculesthat are capable of preventing or inhibiting the interaction of IGF-1with its binding proteins includes ligand inhibitors of IGF-1, asdisclosed in WO 97/39032.

Also suitable for use in the subject methods include IGF-1 pointvariants which bind to IGFBP-1 or IGFBP-3, thus inhibiting theinteraction of endogenous IGF-1 with IGFBPs, which are further describedin U.S. Pat. No. 6,509,443.

Also suitable for use in the subject methods include IGF displacers thatare peptides discovered by phage display that are capable of inhibitingthe interaction of an IGF with any one of its binding proteins, asfurther described in, e.g., U.S. Pat. Nos. 6,420,518; 6,251,865; and6,121,416, all of which are hereby expressly incorporated by referencein their entireties.

Small molecule nonpeptide inhibitors can also release biologicallyactive IGF-1 from the IGF-1/IGFBP-3 complex. For example, isoquinolineanalogues have been found to be effective (See Chen et al., 2001, J MedChem 44:4001-10). Additional compounds can be found using highthroughput screening and the IGFBP Radioligand binding assay asdescribed in Chen et al., 2001.

Other IGF-1 agonists include, but are not limited to; small molecules;synthetic drugs; peptides; polypeptides; proteins; nucleic acids (e.g.,DNA and RNA nucleotides including, but not limited to, antisensenucleotide sequences, triple helices and nucleotide sequences encodingbiologically active proteins, polypeptides or peptides); antibodies;synthetic or natural inorganic molecules; mimetic agents; and syntheticor natural organic molecules.

In addition, the present invention contemplates using gene therapy foradministering IGF-1 to patients. Generally, gene therapy can be used toincrease (or overexpress) IGF-1 levels in the mammal using a recombinantvector to express an IGF-1 gene. Also, gene therapy can be used toexpress a nucleic acid encoding an IGF agonist compound, if it is apeptide. As another example, antisense oligonucleotides can be used toreduce the expression of an IGFBP. Other examples of gene therapy can becontemplated by one of routine skill in the art.

There are two major approaches to introducing the nucleic acid(optionally contained in a vector) into the subject's cells for purposesof gene therapy: in vivo and ex vivo. For in vivo delivery, the nucleicacid is injected directly into the subject, usually at the site whereincreased levels of IGF-1 is required. For ex vivo treatment, thesubject's cells are removed, the nucleic acid is introduced into theseisolated cells and the modified cells are administered to the subjecteither directly or, for example, encapsulated within porous membraneswhich are implanted into the subject. See, e.g. U.S. Pat. Nos. 4,892,538and 5,283,187, both of which are herein expressly incorporated byreference in their entireties.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. A commonly used vector for ex vivodelivery of the gene is a retrovirus.

An example of an in vivo nucleic acid transfer technique includestransfection with viral vectors (such as adenovirus, Herpes simplex Ivirus, or adeno-associated virus) and lipid-based systems (useful lipidsfor lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Chol, forexample). In some situations it is desirable to provide the nucleic acidsource with an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g., capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, and proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,1987, J Biol Chem, 262:4429-4432; and Wagner et al., 1990, Proc NatlAcad Sci USA, 87: 3410-3414. For a review of the currently known genemarking and gene therapy protocols, see Anderson et al., 1992, Science,256: 808-813 and WO 93/25673 and the references cited therein.

Combination Therapy

Combination therapy with IGF-1 and one or more other appropriatereagents, such as those that increase total IGF-1 level in the blood orenhance the effect of the IGF-1, is also contemplated by this invention.In one embodiment, these additional reagents generally allow an excessof blood IGF-1 over the amount of IGFBPs in blood or the IGF-1 to bereleased from IGFBPs, and include growth-promoting agents.

Growth-promoting agents for this purpose include, but are not limitedto, GH secretagogues that promote the release of endogenous GH inmammals to increase concentrations of the IGF in the blood. Examplesinclude TRH, diethylstilbestrol, theophylline, enkephalins, E seriesprostaglandins, peptides of the VIP-secretin-glucagon-GRF family, andother GH secretagogues such as GHRP-6, GHRP-1 as described in U.S. Pat.No. 4,411,890, and benzo-fused lactams such as those disclosed in U.S.Pat. No. 5,206,235. See also, e.g., WO 96/15148 published May 23, 1996.Other growth-promoting agents include GHRPs, GHRHs, GH and theiranalogs. For example, GHRPs are described in WO 95/17422 and WO 95/17423both published Jun. 29, 1995; Bowers, J, 1993, Pediatr Endocrinol,6:21-31; and Schoen et al., 1993, Annual Reports in Medicinal Chemistry,28: 177-186. GHRHs and their analogs are described, for example, in WO96/37514 published Nov. 28, 1996.

The reagent can be co-administered sequentially or simultaneously withIGF-1, and may be administered in the same, higher, or a lower dose thanif used alone depending on such factors as, for example, the type ofreagent used, the purpose for which the reagent and compound are beingused, and clinical considerations. In addition, other means ofmanipulating IGF status, such as regimens of diet or exercise, are alsoconsidered to be combination treatments as part of this invention.

In another embodiment, IGF-1 is appropriately administered together withany one or more of its binding proteins, for example, IGFBP-1, IGFBP-2,IGFBP-3, IGFBP-4, IGFBP-5, or IGFBP-6. Without being bound by amechanism, co-administration of IGF-1 and an IGFBP may provide a greaterresponse than IGF-1 alone by increasing the half-life of IGF-1.

A binding protein suitable for use is IGFBP-3, which is described inU.S. Pat. No. 5,258,287 and by Martin and Baxter, 1986, J Biol Chem,261:38754-8760. This glycosylated IGFBP-3 protein is an acid-stablecomponent of about 53 Kd on a non-reducing SDS-PAGE gel of a 125-150 Kdglycoprotein complex found in human plasma that carries most of theendogenous IGFs and is also regulated by GH.

The administration of the IGF binding protein with IGF-1 may beaccomplished by the method described in U.S. Pat. No. 5,187,151.Briefly, the IGF-1 and IGFBP are administered in effective amounts bysubcutaneous bolus injection in a molar ratio of from about 0.5:1 toabout 3:1, including about 0.75:1 to about 2:1, such as about 1:1.

Subjects Suitable for Treatment

Subjects suitable for treatment with the methods disclosed hereininclude subjects that suffer from IGFD. In general, the IGFD patientpopulation has, for example, the following characteristics: 1) a heightat least about 2 standard deviations (SD) below the normal mean for thecorresponding age and gender, and 2) a blood level of IGF-1 that is atleast 1 SD below normal mean levels. In one embodiment, the presentinvention encompasses methods for treating IGFD children who do not havea blood level of high-affinity growth hormone binding protein that is atleast 2 SDs below normal mean levels, and do not have Laron syndrome. Inanother embodiment, the present invention encompasses methods fortreating IGFD children who do not have a blood level of high-affinitygrowth hormone binding protein that is at least 2 SDs below normal meanlevels, and do not have partial growth hormone insensitivity syndrome(partial GHIS).

In one embodiment, the present invention encompasses methods fortreating IGFD children who have a mean or maximum stimulated blood levelof growth hormone which is at least within the normal range.

In certain embodiments, the subject suffering from IGFD has a height,for example, of at least about 2.0 SD below the normal mean for acorresponding age and gender, at least about 2.5 SD below the normalmean for a corresponding age and gender (i.e., −2.5 SD), or at leastabout 3.0 SD below the normal mean for a corresponding age and gender,usually at least about usually between about 2.0 SD and about 3.0 SDbelow the normal mean for a corresponding age and gender, between about2.5 SD and about 3.0 SD below the normal mean for a corresponding ageand gender, or at least about 3.0 SD below the normal mean for acorresponding age and gender. In certain embodiments, the subjectsuffering from IGFD has a blood level of IGF-1 at least 1 SD below thenormal range for their corresponding age and gender. IGF-1 deficientsubjects can have blood levels of IGF-1 that are, for example, at leastabout 2.0 SD below normal mean levels for a corresponding age andgender, at least about 3.0 SD below normal mean levels for acorresponding age and gender, usually from about 2.0 SD to about 3.0 SDbelow normal mean levels for the corresponding age and gender. An IGFDpatient may also have blood levels of high affinity growth hormonebinding protein less than the normal mean, but not more than 2SD belowthe normal mean. In certain embodiments, the blood level ofhigh-affinity growth hormone binding protein is between normal meanlevels and −0.5 SD below normal mean levels, between normal mean levelsand 0.5 SD below normal mean levels, between 0.5 SD and 1.0 SD belownormal mean levels, between 1.0 SD and 1.5 SD below normal mean levels,or between 1.5 SD and 2.0 SD below normal mean levels.

Short stature patients who will benefit from increased IGF-1 levels canbe identified using routine methods known in the art. IGF-1 levels canbe detected in blood. A genetic abnormality associated with IGF-1 can bedetected using standard genetic assays. A marker for a local IGF-1deficit (such as levels of IGFBP-1) can be detected using routineassays.

Measuring IGF levels in a biological fluid such as a body or blood fluidcan be done by any means, including RIA and ELISA. For example, totalIGF-1 in the blood can be determined by commercially availableradioimmunoassays (Medgenix Diagnostics, Brussels, Belgium; IGF-1 RIAKit, Nichols Institute, San Juan Capistrano, Calif.) especially afterthe extraction of the blood sample using acid ethanol to remove bindingproteins which interfere with the detection of the IGF-1 by competingwith anti-IGF-1 antibody. IGFBP can be measured using commerciallyavailable immunoradiometric assays (IRMAs) for measuring IGFBP-1 andIGFBP-3 (Diagnostic System Laboratories Inc., Webster, Tex.).

Another method involves measuring the level of “free” or active IGF inblood. For example, one method is described in U.S. Pat. No. 5,198,340,herein expressly incorporated by reference in its entirety. Anadditional method is described in U.S. Pat. No. 6,251,865, issued Jun.26, 2001, herein expressly incorporated by reference in its entirety,for detecting endogenous or exogenous IGF bound to an IGF bindingprotein or the amount of a compound that binds to an IGF binding proteinand does not bind to a human IGF receptor bound to an IGF bindingprotein or detecting the level of unbound IGF in a biological fluid.This method comprises: (a) contacting the fluid with 1) a means fordetecting the compound that is specific for the compound (such as afirst antibody specific for epitopes on the compound) attached to asolid-phase carrier, such that in the presence of the compound the IGFbinding sites remain available on the compound for binding to the IGFbinding protein, thereby forming a complex between the means and the IGFbinding protein; and 2) the compound for a period of time sufficient tosaturate all available IGF binding sites on the IGF binding protein,thereby forming a saturated complex; (b) contacting the saturatedcomplex with a detectably labeled second means which is specific for theIGF binding protein (such as a second antibody specific for epitopes onthe IGFBP) which are available for binding when the compound is bound tothe IGF binding protein; and (c) quantitatively analyzing the amount ofthe labeled means bound as a measure of the IGFBP in the biologicalfluid, and therefore as a measure of the amount of bound compound andIGF binding protein, bound IGF and IGF binding protein, or active IGFpresent in the fluid.

U.S. Pat. Nos. 5,593,844 and 5,210,017, herein expressly incorporated byreference in their entireties, disclose a ligand-mediatedimmunofunctional binding protein assay method that can be used toquantitate the amount of IGFBP in a liquid sample by the use ofantibodies, where complex formation takes place between one of thesebinding proteins and the ligand that binds to it.

The quantitative technique mentioned above using antibodies, called theligand-mediated immunofunctional method (LIFA), is described fordetermining the amount of IGFBP by contact with IGF in U.S. Pat. No.5,593,844, herein expressly incorporated by reference in its entirety.

Dosage and Schedule of Administration

Selection of the therapeutically effective dose can be determined (e.g.,via clinical trials) by a skilled artisan, such as a clinician or aphysician, based upon the consideration of several factors which will beknown to one of ordinary skill in the art. Such factors include, forexample, the particular form of IGF-1, and the compound'spharmacokinetic parameters such as bioavailability, metabolism,half-life, and the like, which is established during the developmentprocedures typically employed in obtaining regulatory approval of apharmaceutical compound. Further factors in considering the dose includethe disease or condition to be treated, the benefit to be achieved in asubject, the subject's body mass, the subject's immune status, the routeof administration, whether administration of the compound or combinationtherapeutic agent is acute or chronic, concomitant medications, andother factors known by the skilled artisan to affect the efficacy ofadministered pharmaceutical agents.

The identification and treatment of IGFD as a new condition has directparallels with the identification and treatment of GHD. It has beennoted by others (Drake et al., 2001, Endocrine Reviews 22: 425450) thatit was only the advent of modern neuro-radiological imaging techniquesin 1989 that allowed the diagnosis of GH deficiency in adults to beestablished with certainty. It was this identification of patients withsmall or damaged pituitaries and low IGF-1 levels and low GH levels thatgreatly assisted in establishing a diagnosis of adult GHD. It was alsotherefore only relatively recently that it was recognized that there isa characteristic clinical syndrome associated with failure ofspontaneous GH secretion and that the use of recombinant GH to reversemany of its features has become established.

In terms of how to treat with IGF-1 it is instructive to consider themethods by which GH replacement therapy is practiced. In adults there isno biological marker of GH action that is the equivalent of height orgrowth in a child. Therefore it is difficult to judge the efficacy of GHreplacement in adults. The assessment of optimal GH replacement is madedifficult by the occurrence of side effects if too high doses areadministered. GH treatment is therefore begun at low doses, with dosesthen being increased to the dose that is the final maintenance dose. Itis further very instructive that appropriate GH dosing in adults is bestdetermined by the measurement of blood levels of IGF-1, so as to avoidsupra-physiological levels of IGF-1.

In addition the use of growth hormone antagonists has also beeninstructive. In states of GH excess (such as acromegaly) the current aimof treatment with growth hormone antagonists is to reduce IGF-1 levelsinto the normal range. The measurement of blood levels of IGF-1 has beencharacterized as a sensitive and specific indicator for the presenceacromegaly and the persistence of disease after therapy (Freda, 2003, GHand IGF Research 13:171-184).

There are now normative data on blood levels of IGF-1 that have beenmeasured in many thousands of patients so that IGF-1 standard deviationscores (IGF-1 SDS) have been established (Juul, GH and IGF Research 13,113-170, 2003). Just as in children these normative data are age andgender adjusted to establish the normative range for a subject at agiven age and gender.

It is clearly a parallel argument that appropriate replacement therapyin adults (and in children) is to establish doses of ICE-1 that raiseIGF-1 levels into the age adjusted normal range. There has been muchrecent work to establish the normal range of IGF-1 levels in childrenand adults (Juul, GH and IGF Research 13, 113-170, 2003, hereinexpressly incorporated by reference in its entirety).

In some embodiments, the total pharmaceutically effective amount ofIGF-1 administered parenterally per dose will be in the range of about10 μg/kg/day to about 400 μg/kg/day, including about 20 μg/kg/day toabout 200 μg/kg/day, such as, about 40 μg/kg/day to about 100 μg/kg/day,of subject body weight although, this will be subject to a great deal oftherapeutic discretion. Preferred doses for adults are in the range ofabout 10 μg/kg/day to about 160 μg/kg/day. Other doses of interest foradults are in the range of about 10 μg/kg/day to about 186 μg/kg/day Insome embodiments of particular interest, 20 to 240 μg/kg/day IGF-1 isadministered to the subject. The IGF-1 may be administered by any means,including injections (single or multiple, e.g., 1-4 per day) orinfusions. In certain embodiments, the IGF-1 is administered once ortwice per day by subcutaneous injection. If a slow release formulationis used, typically the dosages used (calculated on a daily basis) willbe less, up to one-half of those described above.

The present invention further provides methods for increasing growthrate using a pharmaceutical composition of IGF-1, and a pharmaceuticallyacceptable carrier. Suitable pharmaceutically acceptable carriersinclude essentially chemically inert and nontoxic pharmaceuticalcompositions that do not interfere with the effectiveness of thebiological activity of the pharmaceutical composition. Examples ofsuitable pharmaceutical carriers include, but are not limited to, salinesolutions, glycerol solutions, ethanol,N-(1(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),diolesylphosphotidylethanolamine (DOPE), and liposomes. Suchpharmaceutical compositions should contain a therapeutically effectiveamount of the compound, together with a suitable amount of carrier so asto provide the form for proper administration to the subject. Theformulation should suit the mode of administration. For example, oraladministration requires enteric coatings to protect the compounds of theinvention from degradation within the gastrointestinal tract. In anotherexample, the compounds of the invention may be administered in aliposomal formulation, particularly for nucleic acids, to shield thecompounds from degradative enzymes, facilitate transport in circulatorysystem, and effect delivery across cell membranes to intracellularsites.

In another embodiment, a pharmaceutical composition comprises a IGF-1protein, and/or one or more therapeutic agents; and a pharmaceuticallyacceptable carrier. In one embodiment, a pharmaceutical composition,comprising a IGF-1 protein, with or without other therapeutic agents;and a pharmaceutically acceptable carrier, is at an effective dose.

The pharmaceutical compositions of the invention can be formulated asneutral or salt forms. Pharmaceutically acceptable salts include thoseformed with free amino groups such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withfree carboxyl groups such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc.

In some embodiments, the composition is formulated in accordance withroutine procedures as a pharmaceutical composition adapted forsubcutaneous injection or intravenous administration to humans.Typically, pharmaceutical compositions for subcutaneous injection orintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lidocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water-free concentrate in a hermetically scaledcontainer such as an ampule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle, bag, or other acceptablecontainer, containing sterile pharmaceutical grade water, saline, orother acceptable diluents. Where the composition is administered byinjection, an ampule of sterile water for injection or saline can beprovided so that the ingredients may be mixed prior to administration.

In certain embodiments, the formulation for IGF-1 is that described inU.S. Pat. No. 5,681,814. This formulation is as follows: about 2 toabout 20 mg/ml of IGF-1, about 2 to about 50 mg/ml of an osmolyte, about1 to about 15 mg/ml of at least one stabilizer, and a buffer (such as anacetic acid salt buffer, or sodium acetate) in an amount such that thecomposition has a pH of about 5 to about 5.5, Optionally, theformulation may also contain a surfactant, preferably in an amount ofabout 1 to about 5 mg/ml, such as about 1 to about 3 mg/ml.

In some embodiments, the osmolyte is an inorganic salt at aconcentration of about 2-10 mg/ml or a sugar alcohol at a concentrationof about 40 to about 50 mg/ml, the stabilizer is benzyl alcohol, phenol,or both, and the buffered solution is an acetic acid salt bufferedsolution. In further embodiments, the osmolyte is an inorganic salt,such as sodium chloride.

In yet further embodiments, the formulation includes about 8 to about 12mg/ml of IGF-1, about 5 to about 6 mg/ml of sodium chloride, benzylalcohol as the stabilizer in an amount of about 8 to about 10 mg/mland/or phenol in an amount of about 2 to about 3 mg/ml, and about 50 mMsodium acetate buffer so that the pH is about 5.4. Optionally, theformulation contains polysorbate as a surfactant in an amount of about 1to about 3 mg/ml.

Pharmaceutical compositions adapted for oral administration may beprovided, for example, as capsules or tablets; as powders or granules;as solutions, syrups or suspensions (in aqueous or non-aqueous liquids);as edible foams or whips; or as emulsions. Tablets or hard gelatinecapsules may comprise, for example, lactose, starch or derivativesthereof, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate, stearic acid or salts thereof. Soft gelatine capsules maycomprise, for example, vegetable oils, waxes, fats, semi-solid, orliquid polyols, etc. Solutions and syrups may comprise, for example,water, polyols and sugars.

An active agent intended for oral administration may be coated with oradmixed with a material (e.g., glyceryl monostearate or glyceryldistearate) that delays disintegration or affects absorption of theactive agent in the gastrointestinal tract. Thus, for example, thesustained release of an active agent may be achieved over many hoursand, if necessary, the active agent can be protected from being degradedwithin the gastrointestinal tract. Taking advantage of the various pHand enzymatic conditions along the gastrointestinal tract,pharmaceutical compositions for oral administration may be formulated tofacilitate release of an active agent at a particular gastrointestinallocation.

Pharmaceutical compositions adapted for parenteral administrationinclude, but are not limited to, aqueous and non-aqueous sterileinjectable solutions or suspensions, which may contain antioxidants,buffers, bacteriostats and solutes that render the pharmaceuticalcompositions substantially isotonic with the blood of an intendedrecipient. Other components that may be present in such pharmaceuticalcompositions include water, alcohols, polyols, glycerine and vegetableoils, for example. Compositions adapted for parenteral administrationmay be presented in unit-dose or multi-dose containers, for example,sealed ampules and vials, and may be stored in a freeze-dried(lyophilized) condition requiring the addition of a sterile liquidcarrier, e.g., sterile saline solution for injections, immediately priorto use. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules and tablets. Such pharmaceuticalcompositions should contain a therapeutically or cosmetically effectiveamount of a compound which increases IGF-1 blood levels, together with asuitable amount of carrier so as to provide the form for properadministration to the subject. The formulation should suit the mode ofadministration.

Pharmaceutical compositions adapted for transdermal administration maybe provided as discrete patches intended to remain in intimate contactwith the epidermis for a prolonged period of time. Pharmaceuticalcompositions adapted for topical administration may be provided as, forexample, ointments, creams, suspensions, lotions, powders, solutions,pastes, gels, sprays, aerosols or oils. A topical ointment or cream ispreferably used for topical administration to the skin, mouth, eye orother external tissues. When formulated in an ointment, the activeingredient may be employed with either a paraffinic or a water-miscibleointment base. Alternatively, the active ingredient may be formulated ina cream with an oil-in-water base or a water-in-oil base.

Pharmaceutical compositions adapted for topical administration to theeye include, for example, eye drops or injectable pharmaceuticalcompositions. In these pharmaceutical compositions, the activeingredient can be dissolved or suspended in a suitable carrier, whichincludes, for example, an aqueous solvent with or withoutcarboxymethylcellulose. Pharmaceutical compositions adapted for topicaladministration in the mouth include, for example, lozenges, pastillesand mouthwashes.

Pharmaceutical compositions adapted for nasal administration maycomprise solid carriers such as powders (preferably having a particlesize in the range of 20 to 500 microns). Powders can be administered inthe manner in which snuff is taken, i.e., by rapid inhalation throughthe nose from a container of powder held close to the nose.Alternatively, pharmaceutical compositions adopted for nasaladministration may comprise liquid carriers such as, for example, nasalsprays or nasal drops. These pharmaceutical compositions may compriseaqueous or oil solutions of the active ingredient. Compositions foradministration by inhalation may be supplied in specially adapteddevices including, but not limited to, pressurized aerosols, nebulizersor insufflators, which can be constructed so as to provide predetermineddosages of the active ingredient.

Pharmaceutical compositions adapted for rectal administration may beprovided as suppositories or enemas. Pharmaceutical compositions adaptedfor vaginal administration may be provided, for example, as pessaries,tampons, creams, gels, pastes, foams or spray formulations.

Suppositories generally contain active ingredients in the range of 0.5%to 10% by weight. Oral formulations preferably contain 10% to 95% activeingredient by weight.

In yet another embodiment, IGF-1 may be administered using long-actingIGF-1 formulations that either delay the clearance of IGF-1 from thesite or cause a slow release of IGF-1 from, e.g., an injection oradministration site. The long-acting formulation that prolongs IGF-1plasma clearance may be in the form of IGF-1 complexed, or covalentlyconjugated (by reversible or irreversible bonding) to a macromoleculesuch as a water-soluble polymer selected from PEG and polypropyleneglycol homopolymers and polyoxyethylene polyols, i.e., those that aresoluble in water at room temperature; See, e.g., U.S. Pat. No.5,824,642, hereby expressly incorporated by reference in its entirety.Alternatively, the IGF-1 may be complexed or bound to a polymer toincrease its circulatory half-life. Examples of polyethylene polyols andpolyoxyethylene polyols useful for this purpose include polyoxyethyleneglycerol, polyethylene glycol, polyoxyethylene sorbitol, polyoxyethyleneglucose, or the like. The glycerol backbone of polyoxyethylene glycerolis the same backbone occurring in, for example, animals and humans inmono-, di-, and triglycerides. The polymer need not have any particularmolecular weight, but it is preferred that the molecular weight bebetween about 3500 and 100,000, more preferably between 5000 and 40,000.Preferably the PEG homopolymer is unsubstituted, but it may also besubstituted at one end with an alkyl group. Preferably, the alkyl groupis a C1-C4 alkyl group, and most preferably a methyl group. Mostpreferably, the polymer is an unsubstituted homopolymer of PEG, amonomethyl-substituted homopolymer of PEG (mPEG), or polyoxyethyleneglycerol (POG) and has a molecular weight of about 5000 to 40,000.

The IGF-1 may also be coupled to a receptor or antibody or antibodyfragment for administration.

Administration of the pharmaceutical compositions of the inventionincludes, but is not limited to, oral, intravenous infusion,subcutaneous injection, intramuscular, topical, depo injection,implantation, time-release mode, intracavitary, intranasal, inhalation,intratumor, intraocular, and controlled release. The pharmaceuticalcompositions of the invention also may be introduced parenterally,transmucosally (e.g., orally), nasally, rectally, intravaginally,sublingually, submucosally, or transdermally. Preferably, administrationis parenteral, i.e., not through the alimentary canal but rather throughsome other route via, for example, intravenous, subcutaneous,intramuscular, intraperitoneal, intraorbital, intracapsular,intraspinal, intrasternal, intra-arterial, or intradermaladministration. The skilled artisan can appreciate the specificadvantages and disadvantages to be considered in choosing a mode ofadministration. Multiple modes of administration are encompassed by theinvention. For example, a IGF-1 protein is administered by subcutaneousinjection, whereas a combination therapeutic agent is administered byintravenous infusion. Moreover, administration of one or more species ofIGF-1 proteins, with or without other therapeutic agents, may occursimultaneously (i.e., co-administration) or sequentially. For example, aIGF-1 protein is first administered to increase sensitivity tosubsequent administration of a second therapeutic agent or therapy. Inanother embodiment, the periods of administration of one or more speciesof IGF-1 protein, with or without other therapeutic agents may overlap.For example, a IGF-1 protein is administered for 7 days, and a secondtherapeutic agent is introduced beginning on the fifth day of IGF-1protein treatment, and treatment with the second therapeutic agentcontinues beyond the 7-day IGF-1 protein treatment. The IGF-1 can alsobe administered intermittently in a cyclical manner as described in U.S.Pat. No. 5,565,428.

In one embodiment, a pharmaceutical composition of the invention isdelivered by a controlled-release or sustained release system. Forexample, the pharmaceutical composition may be administered usingintravenous infusion, an implantable osmotic pump, a transdermal patch,liposomes, or other modes of administration. In one embodiment, a pumpmay be used (See, e.g., Langer, 1990, Science 249:1527-33; Sefton, 1987,CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In anotherembodiment the compound can be delivered in a vesicle, in particular aliposome (See, e.g., Langer, Science 249:1527-33 (1990); Treat et al.,1989, in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-65;Lopez-Berestein, ibid., pp. 317-27 International Patent Publication No.WO 91/04014; U.S. Pat. No. 4,704,355). In another embodiment, polymericmaterials can be used (See, e.g., Medical Applications of ControlledRelease, Langer and Wise (eds.), CRC Press: Boca Raton, Fla., 1974;Controlled Drug Bioavailability, Drug Product Design and Performance,Smolen and Ball (eds.), Wiley: New York (1984); Ranger and Peppas, 1953,J. Macromol. Sci. Rev. Macromol. Chem. 23:61; Levy et al., 1985, Science228:190; During et al, 1989, Ann. Neurol. 25:351; Howard et al., 1989,J. Neurosurg. 71:105). Suitable examples of sustained-releasecompositions include semi-permeable polymer matrices in the form ofshaped articles, e.g., films, or microcapsules. Sustained-releasematrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481),copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (see Sidman etal., 1983, Biopolymers, 22:547-556), poly(2-hydroxyethyl methacrylate)(Langer et al., 1981, J. Biomed Mater Res, 15:167-277), and Langer,1982, Chem Tech, 12:98-105), ethylene vinyl acetate (Langer et al.,supra) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).Sustained-release IGF-1 compositions also include liposomally entrappedIGF-1. Liposomes containing IGF-1 are prepared by methods known per se:DE 3,218,121; Epstein et al., 1985, Proc Natl Acad Sci USA,82:3688-3692; Hwang et al, 1980, Proc Natl Acad Sci USA, 77: 4030-4034;EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat.Appln. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP102,324. Ordinarily, the liposomes are of the small (from or about 200to 800 Angstroms) unilamellar type in which the lipid content is greaterthan about 30 mol percent cholesterol, the selected proportion beingadjusted for the optimal IGF-1 therapy.

In yet another embodiment, a controlled release system can be placed inproximity of the target. For example, a micropump may deliver controlleddoses directly into the brain, thereby requiring only a fraction of thesystemic dose (See, e.g., Goodson, 1984, in Medical Applications ofControlled Release, vol. 2, pp. 115-138). IGF-1 could be delivereddirectly into the peritoneal cavity to preferentially expose visceralfat to drug.

In one embodiment, it may be desirable to administer the pharmaceuticalcomposition of the invention locally to the area in need of treatment,this may be achieved, for example, and not by way of limitation, bylocal infusion during surgery, topical application (e.g., in conjunctionwith a wound dressing after surgery), injection, by means of a catheter,by means of a suppository, or by means of an implant. An implant can beof a porous, non-porous, or gelatinous material, including membranes,such as sialastic membranes, or fibers.

IGF-1 can be administered before, during, and/or after theadministration of one or more therapeutic agents. In yet anotherembodiment, there can be a period of overlap between the administrationof IGF-1 and/or one or more therapeutic agents.

The present invention may be better understood by reference to thefollowing non-limiting Examples, which are provided only as exemplary ofthe invention. The following examples are presented to more fullyillustrate the preferred embodiments of the invention. They should in noway be construed, however, as limiting the broader scope of theinvention.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1 Interrelationships Among Various Measures Related to theGH-IGF Axis

Data from large surveillance studies of the use of GH, such as theNational Cooperative Growth Study (NCGS) can be helpful in determiningpatient populations that will respond to IGF-1 treatment especially withassociated substudies looking at referred but untreated patients andusing centralized assay results. One such substudy of the NCGS (SubstudyVI) noted that short children undergoing hormonal testing were found asa group to have low IGF-1 levels (IGF-1 SDS=−1.7±1.7, mean±SD) despiterelatively normal maximum stimulated GH levels. One of the goals of thissubstudy was to explore the interrelationships among various measuresrelated to the GH-IGF axis, including stimulated GH, GHBP, IGF-1 and IGFbinding protein 3 (IGFBP-3). The data surprisingly showed a substantialproportion of referred short children have primary IGFD, that is, are GHsufficient but IGF-1 deficient.

NCGS substudy VI was designed to evaluate the hormonal basis of shortstature. This study was limited to untreated subjects undergoingevaluation for the hormonal basis of short stature. The protocol-statedobjectives were to 1) identify patients with undetectable or subnormalblood GHBP levels for possible further evaluation, as well as determinethe GHBP levels for subgroups of children with short stature; and 2)define the relationship of GHBP to GH, IGF-1, and IGFBP-3 levels inblood.

Patient Population

Subjects included in the study were evaluated for the hormonal basis oftheir short stature. Only patients for whom baseline specimens weresupplied for GH stimulation test(s), IGF-1, IGFBP-3 and GHBP wereincluded. Patients currently receiving GH therapy were excluded.

Study Design

Upon enrollment, a single plasma sample was collected for themeasurements of GHBP, IGF-1 and IGFBP-3. This was accompanied by up to 8blood samples for measurement of GH levels from one or two standard GHprovocative stimulation tests. An amendment to the protocol provided fora single follow-up blood sample for the repeat measurements of GHBP,IGF-1 and IGFBP-3 at approximately one year after baseline for untreatedsubjects, or after approximately one year of therapy in subjectssubsequently treated with GH.

Laboratory Methods

All specimens were sent to a single laboratory (Nichols Institute) forcentralized determination of hormone levels. Growth hormone was measuredusing the Hybritech immunoradiometric assay (IRMA) using a monoclonalantibody to GH. Such IRMA assays return values that are roughly half thevalue obtained using radioimmunoassay for GH (a GH value of 5 ng/ml inan IRMA roughly equals a value of 10 ng/ml in an RIA). IGF-1 wasmeasured using a radioimmunoassay (RIA, Nichols Institute) followingacid-ethanol extraction. IGFBP-3 was measured by RIA with recombinantstandard and tracer. GHBP was measured by ligand-mediatedimmunofunctional assay (LIFA) (see, e.g., U.S. Pat. No. 5,210,017).

Statistical Analysis

Subjects were included in analyses if enrollment age was between 0 and20 years and all four baseline laboratory measurements were available.Data are presented as mean±standard deviation (SD) except where noted.Data are presented as SD scores (SDS), adjusting for age and genderusing normative data provided for each measurement or assay.

Results

A total of 6447 subjects were evaluated in four cohorts:

1) all 6447 subjects;

2) subjects with height SDS<−2, IGF-1 SDS<−2, and maximum stimulatedGH<10 ng/mL;

3) subjects with height SDS<−2, IGF-1 SDS<−2, and maximum stimulatedGH>10 ng/mL; and

4) subjects with height SDS<−3, IGF-1 SDS<−3, and maximum stimulatedGH>10 ng/mL.

All Subjects

For the entire cohort in the study (n=6447), the mean age at the time ofbaseline diagnostic evaluation was 10.1±4.0 yr, with a mean bone age of8.0±3.8 yr and mean bone age delay=2.2±1.6 yr. The mean height SDS was−2.5±1.1 and mean BMI SDS was −0.5±1.4. At baseline, 68% of subjectswere male and 76% were pre-pubertal. As expected, 77% of subjects had nodefined etiology of their short stature at baseline. Only 75% of thesubjects who were referred for short stature had height SDS<−2 (n=4663),and 87% had a serum IGF-1 that was below normal SDS<0 (normal mean), and39% of these children with short stature, or 1955 children, had a serumIGF-1 of SDS<−2.

The median maximum stimulated (peak) GH level for the 6447 subjects was7.5 μg/L, using the Hybritech assay, equivalent to 15 μg/L byradioimmunoassay (RIA). The mean value for IGF-1 SDS was −1.7±1.7 andfor IGFBP-3 SDS was 1.0±1.6. However, mean GHBP SOS was −0.2±1.2. Thelog peak GH levels were positively correlated with IGF-1 SDS and IGFBP-3SDS (r=0.29, 0.28, respectively) and negatively with GHBP SDS (r=−0.19).A stronger correlation existed between IGF-1 SDS and IGFBP-3 SDS(r=0.65). GHBP SDS was weakly positively correlated with IGF-1 SDS andIGFBP-3 SDS (r=0.15, 0.12, respectively).

Short Subjects with Low IGF-1 and Low GH

Subjects in cohort 2 had height SDS<−2, IGF-1 SDS<−2 and maximumstimulated GH<10 ng/mL. These subjects constitute the “GH deficient”group (n=776 of 1955, or 39% of the short IGF deficient group). In thiscohort, 64% were male and 82% were prepubertal. Mean IGF-1 SDS was−3.8±1.8, with 58% having IGF-1 SDS<−3. This group is the growth hormonedeficient group and are called GHD.

Short Subjects with Low IGF-1 and GH Levels that are at Least Normal

Subjects in the cohort 3 had height SDS<−2, IGF-1 SDS<−2 and maximumstimulated GH>10 ng/mL. These subjects constitute the “IGF deficient/GHsufficient” group (n=1179 of the 1955 patients, or 61% of the short IGFdeficient group). This cohort had a greater percentage of males (71%)compared to cohort 2. Mean IGF-1 SDS was −3.0±0.9, with 41% having IGF-1SDS<−3. Height SOS was <−3 in 39%. This is the group referred to in thisstudy as primary IGFD.

Very Short Subjects with Very Low IGF-1 and GH Levels that are at LeastNormal

Subjects in the cohort 4 had height SDS<−3, IGF-1 SDS<−3 and maximumstimulated GH>10 ng/mL. These subjects constitute a group of extremeshort stature and extreme IGF deficiency (n=212, or 18% of IGFDsubjects). This is the group referred to in this study as severe primaryIGFD.

Discussion

The management of children with marked short stature, despite decades ofstudy, remains a largely subjective undertaking that varies amongcountries, between institutions and even among physicians working at thesame institution. The criteria for using growth-promoting therapies,which currently consist primarily of recombinant human growth hormone(rhGH) and gonadotropin releasing hormone (GnRH) agonists, have includedhormonal, auxologic, radiographic, genetic, ethical and economicfactors. Aside from treating a causal underlying condition (such ashypothyroidism or precocious puberty) or using rhGH for specific,approved indications (such as Turner syndrome), the question ofintervention often comes down to 1) is there a defect in the GH-IGFaxis?; and 2) will final adult height be significantly compromisedwithout treatment?

Tests for GH deficiency usually involve pharmacologic stimuli whichdiffer in their mechanism of stimulating GH release. Only a very smallproportion (about 5-10% of those who are referred to clinics for shortstature) of children with short stature are GH deficient. Howeverseveral studies have suggested that other abnormalities in the GH/IGFsystem might contribute to short stature in a significant number ofnon-GHD patients. For example, in many patients with short stature lowIGF-1 levels are not associated with GH deficiency.

Data from large post-marketing surveillance studies, such as theGenentech National Cooperative Growth Study (NCGS) or Kabi InternationalGrowth Study (KIGS) indicate that a number of non-GH deficient childrenare receiving rhGH therapy, and that they represent a select group ofpatients with a degree of short stature similar to those classified asOH deficient. Many in the field feel that growth-promoting therapy forthese patients is unnecessary on the grounds that they have“normal-variant” short stature, or simply some combination of“constitutional delay of growth and puberty” and “familial shortstature.” However, each of these classifications is dependent ondemonstration that the GH-IGF axis is normal and furthermore, that finaladult height is (or will be) within the mid-parental target range. Onthe contrary, patients being considered for GH treatment typically donot attain their genetic height potential, with or without GH treatmentat standard doses. Furthermore, it appears that many have low IGF-1levels.

Ultimately, deficiency of insulin-like growth factor I (IGF-1), the keymediator of most GH biologic actions, is critical to understandingabnormalities along the GH/IGF axis. Certainly, severe growth hormoneinsensitivity (Laron) syndrome is capable of causing growth failureequal to that seen in severe forms of GH deficiency, due to the similarend-result of profound IGF-1 deficiency. While normal IGF-1 levels areconsidered unusual in confirmed cases of GH deficiency, low IGF-1 levelsare perhaps more common than one would expect in patients who areclearly OH sufficient. In contrast, this study shows that normal OH andabnormal IGF-1 is relatively common. Put another way, IGF-1 deficiencyis relatively common in children who are GH sufficient.

In the absence of malnutrition or liver disease, IGF deficiency in anotherwise healthy individual may be explained by a defect in the GH-IGFaxis. In GH sufficient patients, partial GH insensitivity may exist atthe level of the GH receptor or downstream. In 1-5% of children withso-called idiopathic short stature, demonstrable lesions in theextracellular domain of the GH receptor have been found. Abnormalitiesin GH receptor signal transduction, as measured by tyrosinephosphorylation, have also been reported. Theoretically, other causes ofisolated IGF deficiency or resistance could be due to gene defectsaffecting the Stat5B gene, IGF binding proteins, or the IGF receptor.

The cause of IGF deficiency in most otherwise healthy children is poorlyunderstood. In cases where GH secretion is clearly normal or evenelevated, the cause is necessarily some form of partial OHinsensitivity, although the exact nature of this resistance to GH isunknown in most cases. Patients with ISS enrolled in clinical studies ofGH therapy tend to have low GHBP levels and to respond poorly tostandard doses of rhGH as compared with other short stature groups.However, normal GHBP levels were seen in this substudy of as yetuntreated patients Clearly, whatever selection process physiciansinitiate before placing such children on rhGH therapy, which may haveincluded low IGF-1 levels, delayed bone age, and other factors, resultsin a group with clinical signs of GH resistance.

Recombinant human IGF-1 (rhIGF-1) therapy has thus far been successfullyused in three extremely rare forms of profound IGF deficiency, involvingdefects of the OH receptor gene, the GH gene, or the IGF-1 gene. Thisstudy establishes that there are a substantially larger number ofchildren with unexplained short stature who have some degree of IGF-1deficiency, yet are GH sufficient. As GH deficiency is today treatedwith rhGH replacement therapy, there is a role for rhIGF-1 replacementtherapy in the patients who are IGF deficient.

Example 2 Relationship Between Height and Blood Concentration of IGF-1

The aim of this study was to examine the relationship in adults betweenheight and the blood concentration of IGF-1 and then treat the patientswith rhIGF-1.

Subjects

Individuals who had previously been diagnosed as suffering from Type IIdiabetes mellitus (DM) were selected for study. The 44 subjects were menand women 30 to 70 years of age with a hemoglobin A1c level of greaterthan 8.0%. The subjects were all receiving treatment for hyperglycemiawith oral medication(s) only.

Blood was drawn and height was measured. Total IGF-1 was measured byradioimmunoassay after extracting the sample with acid-ethanol.Hemoglobin A1c levels were also measured. Systolic blood pressure wasalso measured.

The patients were then treated with rhIGF-1, as described in Example 1,at either 20 or 40 micrograms per kilogram twice daily by subcutaneousinjection for 12 weeks.

Results

FIG. 1 shows the positive (r=+0.45) and highly statistically significant(p=0.002) relationship between the height standard deviation score(Height SDS) and the blood concentration of IGF-1 before treatment withrhIGF-1. The IGF-1 values are expressed as logarithms due to theconcentrations not being normally distributed.

Treatment with rhIGF-1 reduced blood glucose levels and reducedhemoglobin Ale levels from 9.9 to 9.1%, a significant fall (p<0.001).

Treatment with rhIGF-1 at 20 micrograms/kg twice daily reduced systolicblood pressure from 140.4 to 130.9 after treatment a fall of 9 mm of Hg,a highly significant fall (p<0.001)

CONCLUSION

An unexpected result was obtained in that the serum concentration ofIGF-1 was positively correlated with the height of the patient. A recentand exhaustive review of the factors affecting blood IGF-1 levels statesthat “[i]n adults, IGF-1 does not correlate with the endogenous GHsecretion . . . ” (Juul, 2003, GH and IGF Research 13:113-170). Thereview goes on to state that “other regulators of IGF-1 in adults mustbe considered.” The factors considered by these authors are “bodycomposition, physical activity, life style habits and changes in sexsteroid levels.” There is no mention of a relationship between heightand IGF-1 levels in adults.

This surprising finding provides the first evidence that many of thefactors such as cardiovascular disease, renal disease, diabetes and bonedisease that have been shown to be associated with short stature areassociated with low IGF-1 levels or IGFD.

In addition the data shows that replacement therapy in these patientshas a significant effect on blood glucose, hemoglobin A1c, and bloodpressure. These are all measures that reflect the diseases associatedwith short stature in adults. Therefore it is clear that replacementtherapy with IGF-1 in these patients is effective- and indicated. IGF-1replacement would be therefore expected to have a significant impact onthe many diseases that result from IGFD and short stature.

Example 3 Administration of rhIGF-1

An animal study was conducted administering rhIGF-1 for the life-time ofanimals to study the long-term effects of rhIGF-1 in normal animals.

It has been shown that the efficacy of GH is limited in humans withnormal GH secretion. Such children with normal GH secretion (so-calledpatients with idiopathic short stature) show very small growth responsesto GH. It might be predicted that the long-term efficacy of rhIGF-1might be limited by such effects as rhIGF-1 causing an acceleration ofbone age, which would cause the epiphyses of the long bones to closeearly which would limit the ability to grow, or of rhIGF-1 inhibiting GHsecretion and thereby having a self-limiting effect on growth.

This Example shows a long-term study in animals that is equivalent tosimilar long-term treatment in growing children. Because the epiphysesof the long bones of rats stay open for a very long period relative totheir life span, it is possible for rats to grow for most of theirlives. The example used a broad range of doses of rhIGF-1 in a verylarge number of animals for a very long period.

Animals

Male and female Crl:CD®(SD)BR VAF/Plus® rats were obtained from thePortage, Mich., facility of Charles River Laboratories, Inc. The animalswere 41 to 44 days old at initiation of treatment. The males weighedfrom 178 to 264 grams, and the females weighed from 131 to 199 grams atinitiation of treatment.

The animals were housed individually (except for the first 3 days ofacclimation when animals were group-housed) in stainless-steel,screen-bottom cages. Some animals were placed in polycarbonate cagesduring the study because of health problems.

Certified rodent diet (#5002 meal, PM® Feeds, Inc.) was provided exceptwhen animals were fasted. The diet was routinely analyzed by themanufacturer for nutritional components and environmental contaminants.

Water was provided ad libitum. Samples of the water are analyzed forspecified microorganisms and environmental contaminants.

Acclimation

Four hundred fifty male rats and four hundred fifty female rats wereacclimated for 14 days (with respect to the male animals) or 15 days(with respect to the female animals) before initiation of treatment. Ingeneral, animals appeared healthy, During acclimation, the animals wereexamined for abnormalities indicative of health problems, an ophthalmicexamination was done, and body weights were recorded for all animalsapproximately 1 week before randomization and at randomization. Foodconsumption was recorded for all animals for approximately 1 week duringacclimation.

Group Designations and Dose Levels

The animals were examined by a laboratory animal veterinarian and foundto be suitable for study inclusion. Selection of animals for the studywas based on clinical observation, body weights, ophthalmicexaminations, and other data as appropriate. Animals were assigned totreatment groups using a blocking procedure designed to achieve bodyweight balance with respect to treatment group. At the time ofrandomization, the weight variation of the animals did not exceed ±2standard deviations of the mean body weight for each gender. Group meanbody weights were analyzed using Levene's test for homogeneity ofvariance at the 5.0% probability level and found to be homogenous.Animals were assigned to the study according to the following design.

TABLE 1 Dose Dose Level Concentration No. of Animals Group(mg/kg/day)^(a) (mg/mL) Male Female Carcinogenicity Animals 1 Vehicle 00 75 75 2 Low (rhIGF-1) 0.25 0.25 75 75 3 Mid 1 (rhIGF-1) 1.0 1.0 75 754 Mid 2 (rhIGF-1) 4.0 4.0 75 75 5 High (rhIGF-1) 10.0 10.0 75 75Satellite Animals 6 Vehicle 0 0 15 15 7 Low (rhIGF-1) 0.25 0.25 15 15 8Mid 1 (rhIGF-1) 1.0 1.0 15 15 9 Mid 2 (rhIGF-1) 4.0 4.0 15 15 10 High(rhIGF-1) 10.0 10.0 15 15 ^(a)The dose volume was 1 mL/kg. Individualdoses were based on the most recently recorded body weights.

Results

Administration of rhIGF-1 caused an increase in body weight gain inmates and females at all dose levels. The magnitude of this effectincreased with increasing dose, although the effect for animals given10.0 mg/kg/day was only slightly greater than that of those given 4.0mg/kg/day. For males, the effect on mean body weight was generallystatistically significant from week 6 throughout the majority of thestudy at the 10.0, 4.0, and 1.0 mg/kg/day dose levels; for males given0.25 mg/kg/day, statistically significant changes were first apparent atweek 22. For females, the effect on mean body weight was generallystatistically significant throughout the majority of the study beginningat week 3 for animals given 10.0 and 4.0 mg/kg/day and week 6 foranimals given 10.0 mg/kg/day. For females given 0.25 mg/kg/day,statistically significant changes generally were noted from week 20 to63. The magnitude of the effect on body weight was marked for animalsgiven 4.0 or 10.0 mg/kg/day. At the beginning of week 69, a time whensurvival for males and females given the high dose was at least 50%,mean body weights for males given 0.25, 1.0, 4.0, or 10.0 mg/kg/day were109%, 116%, 123% and 129% of control values, respectively; for femalesthis was 104%, 113%, 128% and 131% of control values, respectively. Theincreased body weight gain for test material-treated animals wasconsistent with increases in food consumption also noted in thesegroups.

SUMMARY

The results show that in animals with normal GH secretion that rhIGF-1had profound growth promoting activity. In contrast, OH treatment inhumans with normal GH secretion had limited effects, as seen in studieswhere GH is given to children with idiopathic short stature, as opposedto the robust effect of GH treatment in GHD.

Example 4 A Study of Long-Term rhIGF-1 Treatment in Children with ShortStature Due to IGF Deficiency

The objective of this study is to evaluate the efficacy and safety oflong-term replacement therapy with rhIGF-1, in children with shortstature due to IGF deficiency (Pediatric primary IGFD).

Significance to Human Health

Recombinant human IGF-1 has been used in clinical trials to treat themost severely affected cases of Primary IGF Deficiency (Laron Syndromeand several cases of deletion of the human GH gene). The height standarddeviation score of such individuals in the untreated state usuallydeclines with age because of profoundly low linear growth velocities.Doses from 40-120 μg/kg given twice daily by subcutaneous injection havebeen employed. The doses of 40-60 μg/kg have proven marginally effectivewith modest increases in height velocity that were generallyinsufficient to increase the height standard deviation scores, i.e., no“catch-up” growth is observed. Doses in the range of 80-120 μg/kggenerally cause improvements in linear height velocity substantialenough to improve the height standard deviation scores and suchimprovements have been observed to persist for up to 10 years oftreatment. The treatment effect of rhIGF-1 therapy is unknown inpatients with less profound IGFD (e.g., those with heights and serum IGFstandard deviation scores of minus 2 or less). Such subjects suffer froma degree of short stature for which growth hormone therapy is approvedfor subjects with growth hormone deficiency, Turner Syndrome,Intra-uterine growth retardation and Prader-Willi Syndrome. This trialis designed to determine if children with a similar degree of shortstature (height less than −2 SD) and IGFD (blood IGF-1 less than −2 SD)will respond favorably to rhIGF-1 therapy.

Pharmacokinetic analyses of rhIGF-1 in normal adult subjects and insubjects with Types 1 and 2 diabetes strongly suggest that thedisposition of administered rhIGF-1 is greatly influenced by theprevailing concentrations of the IGF binding proteins in serum, mostnotably the concentration of IGFBP-3. A highly significant relationshipexists between serum IGFBP-3 concentrations and the clearance ofadministered rhIGF-1 such that low serum IGFBP-3 concentrations predictrapid clearance of rhIGF-1 and potentially diminish the effect oftreatment.

Children with more modest degrees of Primary IGFD also have less IGFBP-3deficiency than do subjects with Laron Syndrome. Accordingly, a morelimited range of rhIGF-1 doses (50-100 μg/kg, twice daily) is employedin this trial design.

The most extreme form of IGFD is called GHIS, or Laron-type dwarfism(Laron Z et al., 1980, Ann Clin Res 12:269-77; Laron Z et al., 1966, IsrJ Med Sci 2:152-55; Laron Z et al., 1968, Isr J Med Sci 4:883-94), andis transmitted as an autosomal recessive trait. It is most common amongAsiatic Jews and other Middle Eastern people, but occurs sporadically inother ethnic groups. Although molecular heterogeneity of GH-receptordefects have been described (Amselem S et al, 1991, Trends EndocrinolMetab 21:35-40), affected individuals share the clinical characteristicsof severe GH deficiency: they are short, grow at a slow rate, haveimmature facial features and body proportions, and have excess body fat.As in patients with GH deficiency, serum concentrations of IGF-1 arelow. In contrast with GH deficiency, however, serum GH concentrationsare elevated, stimulation of GH secretion produces a supra-normalresponse, and exogenously administered GH does not increase IGF-1 levelsor produce the expected metabolic and growth responses. The basis of theGH resistance in this condition is defective (or absent) GH receptors oncell surfaces. In addition, circulating GH binding proteins, which arehomologous to the extracellular domain of the GH receptor, are oftenundetectable in affected patients. Those in whom the serum GH bindingprotein is found are believed to have a defect in the transmembrane orintracellular domains of the GH receptor or to have a defect in thepost-receptor pathway of GH action (Godowski P et al., 1989, Proc NatlAced Sci (USA) 1989; 86:8083-7; Eshet R et al., 1984, Isr J Med Sci20:8-11).

The form of IGFD addressed in this protocol occurs in children who havedefects in the growth hormone signaling pathway in that their tissuesrespond to growth hormone poorly because they transduce the growthhormone signal very weakly. In addition to short stature, these childrenhave a characteristic biochemical profile that includes high growthhormone levels and inappropriately low circulating levels of IGF-1. Theywould be expected to respond poorly to pharmacologic amounts ofexogenous GH.

The data that rhIGF-1 is an effective form of replacement therapy forsome GHIS patients is based upon in vitro studies with cell linesderived from Laron-type patients and in vivo studies in animals andnormal adult humans. Erythroid progenitor cells and permanentlytransformed T-cell lines derived from patients with IGFD (Laron-type)have been shown to proliferate in response to 1-10 ng/ml of IGF-1 invitro (Geffner M E et al., 1987, J Clin Endocrinol Metab 64:1042-6). Invivo infusion of human IGF-1 stimulates weight gain and linear growth inGH deficient mice (Van Buul-Offers S et al., 1986, Pediatr Res 20:825)and hypophysectomized rats. When infused into insulin-deficient rats,IGF-1 stimulates growth without aggravating hyperglycemia or glycosuria(Schoenle E et al., 1982, Nature 296:252). While slow continuousinfusion of rhIGF-1 seems to be well tolerated (Zapf J et al., 1986, JClin Invest 77:1768), administration of an IV bolus produceshypoglycemia, an anticipated insulin-lice effect (Guler H—P et al.,1987, N Engl J Med 317:137).

Laron and colleagues (Laron Z et al., 1991, Clin Endocr 35:145-50) gaveseven daily subcutaneous injections of recombinant IGF-1 in doses of 120or 150 mg/kg/day to 10 subjects with GHIS (Laron-type). This resulted ina marked rise in serum type III procollagen, and decreases in plasma GH,serum cholesterol, serum SGOT, and serum LDH. A variable response ofplasma insulin was observed, with some patients decreasing their fastinginsulin concentrations while others experienced an increase.

Walker et al. (1991, N Engl J Med 324:1483-8) studied an 8.9-year-oldboy with well-characterized Laron-type IGF-1 deficiency. The child hadphysical and biochemical features typical of the syndrome: severe growthfailure; high serum GH; low IGF-1; absence of GH binding protein;failure to increase IGF-1 in response to short-term administration ofGH; failure to show improved growth during a six-month trial ofGH-therapy. This patient received an 11 day infusion of rhIGF-1(Genentech) and was also observed for 8 days after the infusion. WhereasGH treatment had produced no metabolic effects, the IGF-1 infusioncaused dramatic changes in a variety of metabolic parameters (Walker J Let al., 1991, N Engl J Med 324:1483-8). These results confirmed thatmost of the in vivo effects of GH are mediated through IGF-1 and thatrhIGF-1 replacement can bypass the metabolic resistance to GH. Inaddition, these results suggest that there is a strong likelihood thatrhIGF-1 will produce growth in patients with IGFD due to GH receptordefects. In addition to these metabolic effects, the study showed thatrhIGF-1 infusion could produce fasting hypoglycemia (due to theinsulin-like properties of this peptide) as well as blunt meal-inducedinsulin secretion resulting in postprandial hyperglycemia.

A treatment protocol was developed to determine whether IGF-1 therapycould sustain linear growth in patients with primary IGFD. Patients weremaintained on doses required for optimal growth in the absence of sideeffects. The 120 microgram/kg dose of rhIGF-1 was well tolerated andgave plasma concentrations of IGF-1 in the normal range.

Research Plan

The aim of this protocol was to determine whether long-termadministration of recombinant human insulin-like growth factor I(rhIGF-1), at doses ranging from 80 μg/kg to 120 μg/kg given BID, orTID, by subcutaneous injection to children with primary IGFD is safe andeffective and can restore normal growth and metabolism to children withprimary IGFD.

Subjects:

Patients with growth impairment due to primary IGFD were enrolled.Inclusion criteria included height of at least 2 SD below the normalmean for age; growth rate of less than the 50th percentile for age;plasma IGF-1 at 2 SD below the mean for age; age greater than 2 years;random or stimulated GH levels that are at least normal, which isdefined as a GH level that is greater than or equal to 10 ng/ml.Exclusion criteria included active malignancy or any history ofmalignancy; growth failure due to other reasons; disorders ofgenitourinary, cardiopulmonary, gastrointestinal, or nervous system,other endocrine disorders, nutritional/vitamin deficiencies, orchondrodystrophies; treatment with any corticosteroids or othermedications that influence growth; clinically significant EKGabnormality of a history of clinically significant cardiac arrhythmia.

Methods of Procedure:

Annual Visits:

Anthropometric measurements of height and weight were done by the sameclinician; using standardized equipment. Blood pressure was alsodocumented. Interim history was obtained including assessment for sideeffects of treatment.

Study drug medication records were reviewed. An ECHO was done to assesssize and function of the heart. A renal ultrasound was done to monitorthe size and growth of the kidneys. An audiogram and tympanometry wasdone to assess hearing. DEXA scan was performed to assess bone mineralcontent and body composition.

Treatment:

Subjects received replacement therapy with rhIGF-1 at doses ranging from80-120 micrograms/kg, given subcutaneously BID, or TID, with a maximumtotal dose of 240 micrograms/kg daily. The dose chosen for each patientwas based on patient tolerance and titrated to optimize growth.

If symptoms of hypoglycemia occurred, patients, and parents/guardians ofpatients were instructed to monitor home blood glucose levels using ahome glucose analyzer. Caretakers were instructed to call theinvestigator for readings below 40 or above 200 mg/dl or for symptoms ofhypoglycemia.

Six months after each yearly visit, a Pediatric Endocrinologist examinedthe patient. At this visit the patient was screened for the potentialeffects of treatment, and anthropometric measurements of height andweight were done by the same clinician using standardized equipment.Study drug medication records were also reviewed.

Sample Analysis

Laboratory tests were conducted for serum IGF-1 and GH levels and forCBC, platelet count, serum chemistry and thyroid function tests.

Data Analysis

The growth rate before treatment for these children is approximately2-4.0 cm/yr. Adverse events were addressed and summarized.

Patients were discontinued from the protocol for the following reasons:

-   -   Medical conditions that required study discontinuation.    -   Intercurrent illness, which would, in the judgment of the        Investigator, tend to affect assessments of clinical and mental        status to a significant degree.    -   Patient, parent, or guardian desire to discontinue        participation.    -   Non-compliance with the protocol.

Results and Discussion

In these pediatric patients who were IGFD, treatment with rhIGF-1 causeda significant increase in growth rate.

The five patients shown below in Table 2 were treated with rhIGF-1 forat least one year by twice daily subcutaneous injection of between 80and 120 micrograms per kilogram.

TABLE 2 Patient Characteristics and Growth Rate in IGFD Patients treatedwith rhIGF-1 GH Stimulation Growth Rates Age Test IGF-1 (cm/year)(years) (ng · ml) (ng/ml) Ht SDS Baseline Year 1 2.4 22 25 −3.2 4.2 8.73.4 94.3 47 −4.4 1.5 9.2 4.1 225 25 −4.5 3.0 9.4 7.8 83 115 −2.8 5.1 8.28.6 89 77 −4.9 3.3 9.5 Mean = 3.6 9.0

GH Levels

The growth hormone levels measured in these patients after the GHstimulation test were all above the level designated as normal (10ng/ml). Therefore, all the patients in the study were GH sufficient.

IGF-1 Levels

The levels of IGF-1 were compared with the normative data sets from 2sources to estimate the IGF-1 SDS values in the 5 patients above. TheIGF-1 SDS values were within the normal range (an SDS value less than 2below the normal mean) for the above patients using at least one of the2 normative data. These patients therefore can be designated as havingPediatric primary IGFD or severe Pediatric IGFD, depending for somepatients which of the normative datasets are used to calculated theIGF-1 SDS values.

Growth Rates

The baseline height SDS score in these patients were all less than 2below the mean. Therefore, these patients can also be designated assuffering from IGFD or severe IGF. The baseline growth rate of thepatients averaged 3.6 cm per year. When the patients were treated withrhIGF-1 their growth rates were increased to on average 9.0 cm per year.The increase in growth rate of 5.4 cm is a clinically significantincrease.

The data therefore shows that treatment with rhIGF-1 in Pediatricprimary IGFD patients and severe Pediatric primary IGFD patientsaccelerates growth rates.

All references cited herein are specifically incorporated by referenceas if fully set forth herein.

Having hereinabove disclosed exemplary embodiments of the presentinvention, those skilled in the art will recognize that this disclosureis only exemplary such that various alternatives, adaptations, andmodifications are within the scope of the invention, and arecontemplated by the Applicant. Accordingly, the present invention is notlimited to the specific embodiments as illustrated above, but is definedby the following claims.

1.-33. (canceled)
 34. A method for treating a human pediatric subjecthaving secondary insulin-like growth factor-1 deficiency (IGFD), themethod comprising; administering to the human pediatric subject aneffective amount of insulin like growth factor-1 (IGF-1) and growthhormone (GH), wherein the subject is characterized as follows: a) at thetime of treatment or prior to initial treatment with IGF-1, has or had aheight at least about 2 standard deviations (SD) below the normal meanheight for a human pediatric subject of the same age and gender, b) atthe time of treatment or prior to initial treatment with IGF-1, has orhad a blood level of IGF-1 at least about 1 SD below normal mean levelsfor a human pediatric subject of the same age and gender, and c) thehuman pediatric subject does not have Laron syndrome or partial growthhormone insensitivity syndrome, wherein said administering is effectiveto treat secondary IGFD in the human pediatric subject.
 35. The methodof claim 34, wherein the subject is further characterized as having ablood level of growth hormone binding protein (GHBP) which is at leastnormal for a subject of the same age and gender.
 36. The method of claim34, wherein said administering alleviates at least one symptom of IGFD.37. The method of claim 34, wherein said administering provides for anincrease in growth rate or height.
 38. The method of claim 34, whereinthe subject has a blood level of IGF-1 that is at least about 2.0 SDbelow normal mean levels for a subject of the same age and gender. 39.The method of claim 34, wherein IGF-1 is administered in a dose of about20 to 240 μg/kg/day.
 40. The method of claim 39, wherein said IGF-1 isadministered subcutaneously.
 41. The method of claim 34, wherein theIGF-1 is recombinant human IGF-1.
 42. The method of claim 34, whereinthe IGF-1 is an IGF-1 variant.
 43. The method of claim 34, wherein theIGF-1 is a variant IGF-1 lacking up to five amino acids from theN-terminus, compared to native IGF-1.
 44. A method for treating a humanadult subject having secondary insulin-like growth factor-1 deficiency(IGFD) comprising; administering to the human adult subject an effectiveamount of insulin like growth factor-1 (IGF-1) and growth hormone (GH),wherein the subject is characterized as follows: a) at the time oftreatment or prior to initial treatment with IGF-1, has or had a heightat least about 2 standard deviations (SD) below a normal mean for ahuman adult subject of the same age and gender, b) at the time oftreatment or prior to initial treatment with IGF-1, has or had a bloodlevel of IGF-1 at least about 1 SD below normal mean levels for a humanadult subject of the same age and gender, and c) the human adult subjectdoes not have Laron syndrome or partial growth hormone insensitivitysyndrome, wherein said administering provides for treatment of IGFD inthe human adult subject.
 45. The method of claim 44, wherein the subjectis further characterized as having a blood level of growth hormonebinding protein (GHBP) which is at least normal for a subject of thesame age and gender.
 46. The method of claim 44, wherein saidadministering alleviates at least one symptom of IGFD.
 47. The method ofclaim 44, wherein the subject has a blood level of IGF-1 that is atleast about 2.0 SD below normal mean levels for a subject of the sameage and gender.
 48. The method of claim 44, wherein IGF-1 isadministered in a dose of about 20 to 240 μg/kg/day.
 49. The method ofclaim 48, wherein said IGF-1 is administered subcutaneously.
 50. Themethod of claim 44, wherein the IGF-1 is recombinant human IGF-1. 51.The method of claim 44, wherein the IGF-1 is an IGF-1 variant.
 52. Themethod of claim 44, wherein the IGF-1 is a variant IGF-1 lacking up tofive amino acids from the N-terminus, compared to native IGF-1.
 53. Amethod for achieving at least normal insulin-like growth factor-1(IGF-1) levels for age and gender in a human subject having secondaryinsulin-like growth factor-1 deficiency (IGFD), comprising administeringan effective amount of insulin-like growth factor (IGF-1) and growthhormone (GH) to the human subject, wherein the human subject ischaracterized as follows: a) at the time of treatment or prior toinitial treatment with IGF-1, has or had a height at least about 2standard deviations (SD) below a normal mean for a human subject of thesame age and gender, and b) at the time of treatment or prior to initialtreatment with IGF-1, has or had a blood level of IGF-1 at least about 1SD below normal mean levels for a human subject of the same age andgender, wherein the human subject does not have Laron syndrome orpartial growth hormone insensitivity syndrome, and wherein saidadministering achieves normal blood IGF-1 levels for age and gender inthe human subject.
 54. The method of claim 53, wherein the subject isfurther characterized as having a blood level of growth hormone bindingprotein (GHBP) which is at least normal for a subject of the same ageand gender.
 55. The method of claim 53, wherein the subject has a bloodlevel of IGF-1 that is at least about 2.0 SD below normal mean levelsfor a subject of the same age and gender.
 56. The method of claim 53,wherein IGF-1 is administered in a dose of about 20 to 240 μg/kg/day.57. The method of claim 56, wherein said IGF-1 is administeredsubcutaneously.
 58. The method of claim 53, wherein the IGF-1 isrecombinant human IGF-1.
 59. The method of claim 53, wherein the IGF-1is an IGF-1 variant.
 60. The method of claim 53, wherein the IGF-1 is avariant IGF-1 lacking up to five amino acids from the N-terminus,compared to native IGF-1.